ATF4/NUPR1 axis promotes cancer cell survival and mediates immunosuppression in clear cell renal cell carcinoma

ATF4/NUPR1 axis promotes cancer cell survival and mediates immunosuppression in clear cell renal cell carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ATF4/NUPR1 axis promotes cancer cell survival and mediates immunosuppression in clear cell renal cell carcinoma Yongliang Lu, Weihao Chen, Yundong Xuan, Xiubin Li, Shengpan Wu, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3870049/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Cancer cells encounter unavoidable stress during tumor growth. The stress-induced transcription factor, activating transcription factor 4 (ATF4), has been reported to upregulate various adaptive genes involved in salvage pathways to alleviate stress and promote tumor progression. However, this effect is unknown in clear cell renal cell carcinoma (ccRCC). In this study, we found that ATF4 expression was remarkably upregulated in tumor tissues and associated with poor ccRCC outcomes. ATF4 depletion significantly impaired ccRCC cell proliferation, migration, and invasion in vitro and in vivo by inhibiting the AKT/mTOR and epithelial–mesenchymal transition (EMT)-related signaling pathway. RNA sequencing and functional studies identified nuclear protein 1 (NUPR1) as a key downstream target of ATF4 for repressing ferroptosis and promoting ccRCC cell survival. In addition, targeting ATF4 or pharmacological inhibition using NUPR1 inhibitor ZZW115 promoted antitumor immunity in syngeneic graft mouse models, represented by increased infiltration of CD4 + and CD8 + T cells. Furthermore, ZZW115 could improve the response to the PD-1 immune checkpoint blockade. The results demonstrate that the ATF4/NUPR1 signaling axis promotes ccRCC survival and facilitates tumor-mediated immunosuppression, providing a set of potential targets and prognostic indicators for ccRCC patients. ccRCC ATF4 NUPR1 Ferroptosis Immunosuppression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Renal cell carcinoma (RCC) is the sixth leading cause of cancer-related deaths globally, with a slight increase in associated morbidity and mortality in recent years ( 1 , 2 ) . Approximately 30% of RCC patients are initially diagnosed with advanced or metastatic disease due to occult early symptoms and a lack of markers ( 3 ) , and approximately 75–80% of the pathological RCC types are clear cell renal cell carcinoma (ccRCC) ( 4 ) . Although surgery is potentially curative for ccRCC when detected at an early stage, postoperative recurrence remains common, and tyrosine kinase inhibitors and immunotherapy for advanced ccRCC remain unsatisfactory owing to drug resistance ( 5 ) . Consequently, it is crucial to acquire in-depth insights into the molecular mechanisms underlying ccRCC occurrence and development to identify new targets and develop therapies for its treatment. Rapidly growing tumors with excessive proliferation escalate the demand for protein synthesis, which contributes to endoplasmic reticulum (ER) stress ( 6 – 8 ) , and cells in the tumor core suffer from a lack of oxygen and nutrients due to insufficient and defective vascularization. Consequently, sets of adaptive genes are activated in response to these challenges ( 9 ) , and there is a common downstream effector protein, ATF4, that is frequently upregulated in cancer cells. It controls the expression of target genes, most of which are involved in various salvage pathways that relieve stress and promote cell survival ( 10 ) . Recent studies have revealed that ATF4 is required for distinct steps to increase amino acid availability in the autophagic pathway, and it may enhance mTOR activity by upregulating adaptive genes ( 11 , 12 ) . It is thus evident that ATF4 plays a pivotal role in alleviating stress during rapid tumor growth. Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation ( 13 ) . Various metabolic processes, such as those associated with iron and amino acids, have been suggested to be related to ferroptosis ( 14 ) . ATF4 is known to be an adaptive regulator of cancer cell responses to various stresses, including ER stress during ferroptosis by erastin ( 15 , 16 ) . Moreover, a recent study demonstrated that ferroptosis is related to the tumor microenvironment (TME) features of ccRCC, and it may thus play a key role in the response to immunotherapy ( 17 ) . However, the functions and regulatory mechanisms of ATF4 remain largely unknown in ccRCC. Therefore, this study aimed to investigate the role of ATF4 in ccRCC, especially its function in ferroptosis and tumor immunology. 2. Materials and Methods Cell culture, reagents, and transfection The human renal proximal tubular epithelial cell lines (HEK293T and HKC), ccRCC cell lines (A498 ACHN, Caki-1, 786-O, SN12), and murine RCC cell line (RenCa) were purchased in September 2015 from the National Platform of Experimental Cell Resources for Sci-Tech (Beijing, China) and authenticated in January 2019 using a previously described method ( 18 ) . All cell lines were cultured in the recommended medium supplemented with 10% fetal bovine serum (FBS; Evergreen Co. Ltd., China) and 1% penicillin–streptomycin at 37°C in 5% CO 2 under mycoplasma-free conditions. pLKO-shATF4 plasmids with short hairpin RNAs (shRNAs) targeting human and murine ATF4 were designed by Invitrogen. The target shATF4 sequences in humans and mice are enumerated in Supplementary Table S1 . Plasmid construction and transfection were performed using a method described previously ( 18 ) . Chemical reagents, including erastin (CAS No. 571203-78-6), ferrostatin-1 (CAS No. 347174-05-4), ZZW115 (CAS No. 801991-87-7), and anti-PD1 (CAS No. 946414-94-4) were purchased from MedChem Express (Shanghai, China). Immunohistochemistry (IHC) and analysis The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Research Ethics Committee of the PLA General Hospital. A total of 199 pairs of ccRCC and normal tissue specimens were included from previously constructed tissue microarrays (TMAs) in our laboratory. Immunostaining of the TMA and xenograft tumor tissues was performed as previously described ( 19 ) . The primary antibodies included rabbit anti-ATF4 (Proteintech; 10835-1-AP), rabbit anti-NUPR1 (Proteintech; 15056-1-AP), rabbit anti-CD4 (Abcam; ab183685), rabbit anti-CD8 alpha (Abcam; ab217344), rabbit anti-cleaved caspase-3 (Cell Signaling Technology; #9661), mouse anti-granzyme B (Santa Cruz; sc-8022), and rabbit anti-Ki-67 (Cell Signaling Technology; #9129). TMA samples were classified according to the intensity of ATF4 or NUPR1 staining, and their expressions were scored as follows: 0, negative; 1, very weak; 2, moderate; and 3, strong. Cases with scores of 0/1 and 2/3 were defined as the ATF4 or NUPR1 low- and high-expression groups, respectively. Tumor-infiltrating immune cells were evaluated as previously described ( 20 ) . Real-time PCR In accordance with the manufacturer’s protocol, total RNA was extracted from cells using the PARISTM Kit (Applied Biosystems) and then reverse-transcribed to obtain cDNA. With the Applied Biosystems 7500 Detection system, real-time PCR was performed. Relative mRNA levels were normalized to human peptidylprolyl isomerase A (PPIA) and calculated using the 2 −ΔΔC T method ( 21 ) . Supplementary Tables S2 and S3 specify the primer sequences and reaction conditions of mixtures for real-time PCR. Immunoblotting and Antibody Western blot was performed as described previously ( 18 ) . Primary antibodies included rabbit anti-ATF4 (Proteintech; 10835-1-AP), rabbit anti-mTOR (Cell Signaling Technology; #2983S), rabbit anti-phospho-mTOR (Ser2448) (D9C2) XP® (Cell Signaling Technology; #5536S), rabbit anti-S6K (Proteintech; 14485-1-AP), rabbit anti-phospho-S6K (Proteintech; 28735-1-AP), rabbit anti-4EBP1 (Cell Signaling Technology; #9644S), rabbit anti-phospho-4EBP1 (Cell Signaling Technology; #2855S), rabbit anti-N-cadherin (Proteintech; 22018-1-AP), rabbit anti-E-cadherin ((Proteintech; 20874-1-AP), rabbit anti-vimentin (Proteintech; 10366-1-AP), rabbit anti-cleaved caspase-3 (Cell Signaling Technology; #9661), rabbit-anti-BCL2 (Cell Signaling Technology; #3498S), rabbit anti-AKT (Cell Signaling Technology; #4685S), rabbit anti-phospho-Akt (Ser473) (D9E) XP® (Cell Signaling Technology; #4060S), and rabbit anti-NUPR1 (Proteintech; 15056-1-AP). Flow cytometry for cell apoptosis analysis and lipid ROS analysis An Annexin V-FITC/PI kit (Abbkine Scientific Co., Ltd, KTA0002) was used to investigate the apoptotic effect of ATF4 deletion and the NUPR1 inhibitor. Lipid ROS were stained with C11-BODIPY 581/591 (RM02821, ABclonal Technology Co., Ltd, Wuhan, China) and analyzed using flow cytometry and fluorescence microscopy in accordance with the manufacturer’s protocols. The mean fluorescence intensity was estimated using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). All samples were analyzed in triplicate, and the data were analyzed using FlowJo version 10 software. Fluorescence imaging was performed using TissueFAXS (TissueGnostics GmbH, Vienna, Austria) with a Zeiss Axio Imager Z2 microscope system. Cell immunofluorescence: Terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and Fe 2+ level assays Cells were placed in culture dishes overnight for adhesion, and the relevant treatment was then applied. The TUNEL Apoptosis Detection Kit (Green Fluorescence; Abbkine Scientific Co., Ltd, KTA2010) or FerroOrange (Dojindo Laboratories, Beijing, China) was used in accordance with the manufacturer’s instructions to determine cellular apoptosis or Fe 2+ levels. Fluorescence images were obtained using TissueFAXS (TissueGnostics GmbH, Vienna, Austria). A quantitative analysis of the mean fluorescence signal intensity was conducted using the ImageJ software. All samples were analyzed in triplicate. Animal modeling and treatment The animal study protocol was approved by the Institutional Animal Care and Use Committee of the PLA General Hospital. Tumor cells (1 × 10 6 ) with PBS or Matrigel (Corning) were implanted into the kidneys of male BALB/c (nude) mice (Vital River, Beijing, aged 4–5 weeks) for the xenograft model or syngeneic graft. After three weeks, the mice were sacrificed, and bioluminescent imaging was performed. For the anti-PD1 treatment, the mice received 200 µg of anti-PD1 via intraperitoneal injection on days 8, 11, 14, 17, and 20, and for the NUPR1 inhibitor ZZW115 treatment, mice received 1 mg/kg ZZW115 via intraperitoneal injection daily (treatment on days 8–21). The maximal tumor size/burden permitted by our institutional review board is mean tumor diameter 20 mm and 15% of body weight in adult mice (~ 25 g). The maximal tumor size/burden in our study was not exceeded. Statistical analysis Data are presented herein as mean ± standard error of measurement (SEM) from at least three independent experiments. ANOVA, Mann–Whitney U, or Student’s t-test was used when appropriate to perform comparisons. The Kaplan–Meier method, log-rank test, and multivariate Cox proportional hazard models were used to detect survival differences and identify independent prognostic factors. Statistical analyses were carried out using SPSS 22.0 (SPSS Inc., USA). The level of significance was set at *p < .05, **p < .01, ***p < .001, and ****p < .0001. 3. Results 3.1. ATF4 expression is upregulated in ccRCC and associated with poor outcomes To evaluate ATF4 expression and its potential clinical role in ccRCC, we investigated the mRNA expression of ATF4 in ccRCC by analyzing the Cancer Genome Atlas (TCGA) data, indicating that ATF4 expression was significantly higher in ccRCC tumor tissues than in normal tissues (Fig. 1 a). ATF4 expression was also associated with prognosis, and the overall survival (OS) probability of the ATF4 high-expression group was found to be significantly higher than that of the ATF4 low-expression group (Fig. 1 b). Western blot experiments on five paired ccRCC tissues and normal tissues from patients revealed that ATF4 was upregulated in tumor tissues (Fig. 1 c). We then evaluated ATF4 expression in ccRCC by conducting an IHC staining analysis of a TMA consisting of 199 ccRCC and 143 normal tissue samples. A variable ATF4 staining intensity was detected in ccRCC tumor tissues using TMAs (Fig. 1 d), and patients were divided into low- and high-expression groups based on the IHC score according to ATF4 expression level. The IHC score for ATF4 in tumor tissues was higher than in normal tissues (Fig. 1 e). The Kaplan–Meier analysis revealed that ATF4 high expression was significantly correlated with a shorter OS (p = 0.0043; Fig. 1 f). Consistently, ATF4 expression also correlated with various features of tumor progression, including T stage, M stage, and histological grade. Quantitative analyses based on IHC scores indicated that high ATF4 expression was significantly associated with advanced T stage (T3 and T4), M1 stage, and advanced tumor grade (III and IV; Fig. 1 g, Table 1 ). Taken together, these findings suggest that ATF4 expression is upregulated in ccRCC and associated with poor outcomes. Table 1 Association of ATF4 expression with clinical and pathological characteristics of 199 ccRCC patients in TMAs. 3.2. ATF4 knockdown attenuates ccRCC cell proliferation, migration, and invasion and induces apoptosis To investigate the functional role of ATF4 in ccRCC cells, we first examined the protein expression of ATF4 in normal renal epithelial cells (HEK293T and HKC) and ccRCC cell lines (A498, ACHN, Caki-1, 786-O, and SN12). The results revealed that a higher ATF4 expression was detected in ccRCC cell lines compared with HEK293T and HKC cell lines (Fig. 2 a). Among these cell lines, the top two highest expressions of ATF4 were observed in the SN12 and 786-O cell lines, which were then used for further studies in vitro. Two lentiviral vectors with shRNA against ATF4 (shATF4) were constructed, the efficacy of which was confirmed by Western blot on day 4 after lentivirus infection (Fig. 2 b). ATF4 depletion was found to inhibit cell proliferation by MTT assay (Fig. 2 c) and impair migration and invasion abilities by transwell assay (Fig. 2 d). Flow cytometry (Fig. 2 e) analyses consistently revealed that ATF4 knockdown induced remarkable early apoptosis in both 786-O and SN12 cells. We then detected several markers related to mTOR, EMT, and apoptosis by Western blot. ATF4 knockdown was found to exert a remarkable effect on the phosphorylation of S6 and 4EBP1, a readout of mTORC1 signaling. The increased expression of E-cadherin was observed in 786-O cells infected with shATF4, together with the concomitant decreased expression of N-cadherin and vimentin. In addition, shATF4-treated cells exhibited an increase in cleaved caspase-3 activity and a reduction in Bcl-2 expression compared with the NC group (Fig. 2 f). In vivo, the orthotopic implantation model in BALB/c nude mice was established by transplanting Luc-labelled SN12 cells, stably transfected with NC or shATF4 under the right kidney capsule. On day 21 after injection, tumor signals by bioluminescent imaging and tumor weight in the shATF4 group were significantly lower than in the NC group (Supplementary Figure S1 , Fig. 2 g). Consistent with the in vitro results, IHC for tumor tissues showed higher Ki-67 and cleaved caspase-3 expressions in shATF4 group (Fig. 2 h). Altogether, these findings suggest that ATF4 may serve a pro-survival function by promoting the mTOR signaling pathway and EMT and inhibiting apoptosis in ccRCC. 3.3. ATF4 deficiency downregulates NUPR1 expression in ccRCC To verify the molecular mechanisms or signaling pathways involved in ATF4-mediated pro-survival in ccRCC, an RNA-seq analysis of 786-O cells was performed after ATF4 knockdown. A heatmap analysis of differentially expressed genes (DEGs, |fold change|>2) showed overall changes (Fig. 3 a). A Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis and gene ontology (GO) enrichment analysis were performed to examine the functional role of ATF4 in ccRCC, which indicated that ATF4 may participate in the PI3K/AKT signaling pathway in ccRCC (Fig. 3 b). Target genes transcriptionally regulated by ATF4 were acquired based on chromatin immunoprecipitation (ChIP) results from other studies in the database. A Venn diagram of target genes and DEGs was drawn to screen the potential genes, and 75 potential genes were identified and shown in the DEG volcano plot (Fig. 3 c), among which NUPR1 showed the most remarkable fold change (Fig. 3 d; http://www.bioinformatics.com.cn ). The expression correlation between ATF4 and NUPR1 was identified based on ccRCC cases in the TCGA dataset (Supplementary Figure S2a). To verify whether NUPR1 is regulated by ATF4 in ccRCC, the relative mRNA and protein levels of NUPR1 were measured by qRT-PCR and Western blot in 786-O cells and SN12 cells. NUPR1 was found to be downregulated in the shATF4 group (Figs. 3 e and 3 f). IHC showed the same results in tumor tissues (Supplementary Figure S2b). Moreover, the linear regression analysis results based on the IHC score for 199 ccRCC tissues by TMAs demonstrated a significant correlation between NUPR1 and ATF4 expressions (Fig. 3 g). Overall, our data suggest that ATF4 regulates NUPR1 expression during ccRCC progression. 3.4. NUPR1 expression is correlated with an advanced stage and prognosis in patients with ccRCC NUPR1 is also known as p8 and is a candidate of metastasis 1 (COM1) and is overexpressed in various cancers in response to several cellular stress factors. Remarkably, multiple types of cancer can be prevented by genetically inactivating NUPR1 ( 22 – 30 ) . Mechanistically, NUPR1 may promote tumor progression by activating the PI3K/AKT signaling pathway, and it is also a critical repressor of ferroptosis that blocks ferroptotic cell death by diminishing iron accumulation and subsequent oxidative damage via LCN2 expression ( 31 – 33 ) . The data from TCGA revealed that NUPR1 mRNA was significantly upregulated in ccRCC compared with normal tissues and correlated with the tumor clinical stage (Fig. 4 a). The OS probability of the NUPR1 high-expression group was significantly lower than the NUPR1 low- and medium-expression groups (Fig. 4 b). NUPR1 expression was detected in 199 ccRCC and 143 normal tissues from TMAs via IHC staining. These patients were divided into NUPR1 low- and high-expression groups based on their IHC score according to the variable staining intensity (Fig. 4 c). The IHC score for NUPR1 in tumor tissues was markedly higher than the score corresponding to normal tissues (Fig. 4 d). NUPR1 expression was also found to be significantly associated with the T stage (Fig. 4 e). The Kaplan–Meier analysis showed that the NUPR1 high-expression group was significantly correlated with shorter OS (Fig. 4 f). In summary, these findings disclose that NUPR1 expression is upregulated in ccRCC and associated with poor outcomes. 3.5. NUPR1 inhibitor ZZW115 reduces cell proliferation and metastasis and induces apoptosis and ferroptosis The NUPR1 inhibitor ZZW115 binds strongly the nuclear localization signal (NLS) of NUPR1, which prevents NUPR1 from translocating to the nucleus. ZZW115 acts mechanistically by mimicking NUPR1 inactivation ( 34 ) . To explore whether NUPR1 acts as a tumor promoter and if it can serve as a potential therapeutic target in ccRCC, MTT, and transwell assay were conducted to examine the effect of ZZW115 on ccRCC cell growth, migration, and invasion. In vitro experiments revealed that ZZW115 inhibited cell growth and metastasis and increased the cell death of 786-O and SN12 cells in a dose-dependent manner (Figs. 5 a, 5 b, and Supplementary Figure S3). TUNEL imaging and flow cytometry showed significantly more apoptotic cells in the ZZW115 group than in the control group (Figs. 5 d and 5 c). The Western blot analysis showed lower expression levels of p-AKT/p-mTOR, N-cad, and vimentin in ZZW115-treated 786-O cells than DMSO and increased expressions of E-cad and cleaved caspase-3. In addition, the ZZW115 treatment induced the accumulation of lipid hydroperoxide, suggesting a promotion of ferroptosis (Fig. 5 e). Altogether, these findings suggest that ATF4 knockdown may suppress tumor progression by decreasing NUPR1 expression. 3.6. ATF4 knockdown induces ferroptosis by targeting NUPR1 NUPR1 is a critical repressor of ferroptosis, and an NUPR1 inhibitor induces ferroptosis in ccRCC. It was hypothesized that ATF4 knockdown could induce ferroptosis by downregulating NUPR1. Erastin dose-dependently induced cell death in ccRCC cells, which was partially rescued by the ferroptosis inhibitor ferrostatin-1 (Supplementary Figure S4a). Previous studies indicated that ATF4 could also have interaction with erastin-induced stress ( 15 , 16 ) . Indeed, erastin was found to dose-dependently induce feedback upregulation of ATF4 expression, suggesting that ATF4 may be associated with ferroptosis (Fig. 6 a). ATF4 deletion simultaneously downregulated erastin-induced NUPR1 protein expression (Fig. 6 b) and increased erastin-induced lipid peroxidation and cell death (Supplementary Figures S4b and S4c). Treatment with shATF4 induced an evident decrease in mitochondria volume, a distinct reduction (or disappearance) of mitochondrial cristae, and an increase in membrane density, which are described as morphological features in ferroptotic cells (Fig. 6 c). Flow cytometry analysis demonstrated a remarkable increase in lipid peroxidation in the shATF4 group compared with the NC group, which was rescued by the ferroptosis inhibitor ferrostatin-1 (Figs. 6 d and 6 e). To examine whether ATF4 deletion increased Fe 2+ levels, an analysis was conducted with the fluorescence probe FerroOrange. The results revealed a significant increase in the relative intracellular Fe 2+ fluorescence intensity in shATF4-transfected 786-O and Caki-1 cells compared with the NC group (Figs. 6 f and S4d). Likewise, the NUPR1 inhibitor ZZW115 increased the intracellular Fe 2+ levels in Caki-1 cells (Figure S4e). Ferrostatin-1 increased cell viability compared with DMSO in the shATF4 group, partially rescuing cell death by ATF4 deletion (Fig. 6 g). Collectively, these findings indicate that ATF4 knockdown induces ferroptosis by increasing intracellular Fe 2+ levels by downregulating NUPR1 expression in ccRCC cells. 3.7. ATF4 depletion increases the infiltration of CD4 + and CD8 + T cells and promotes antitumor immunity in vivo To investigate whether ATF4 deletion promotes an antitumor immune response to inhibit tumor growth and metastasis, murine shATF4 (shATF4-M) for RenCa cells was generated and validated by Western blot and proliferation assays on day 4 after lentivirus infection (Supplementary Figures S5a and S5b). An orthotopic implantation model was established by transplanting RenCa cells with negative control shRNA or shATF4-M into the kidney (Supplementary Figures S5c and S5d). ATF4 knockdown significantly reduced the tumor weight and lung metastasis (Figs. 7 a and 7 b). Genomic analysis has proven that ferroptosis is associated with programmed death-ligand 1 (PD-L1) expression, immune cell infiltration, and immunotherapy sensitivity ( 17 , 35 ) . Although ferroptosis may be involved in a diverse and complex TME, it remains largely unclear whether ATF4 influences TME and immunotherapy in ccRCC. The TME alterations were detected in the BALB/c mice, and intratumor infiltration of CD4 − , CD8 − , and granzyme B + cells remarkably increased in tumors from the shATF4 group (Fig. 7 c). The same tendency was observed in tumor tissues from TMAs (Fig. 7 d). Recent studies have proven that CD8 + T cells suppress tumor growth by inducing ferroptosis. Targeting tumor ferroptosis-associated metabolism may thus improve the efficacy of cancer immunotherapy. A synergistic effect has been reported between PD-L1 blockade antibodies and ferroptosis activators, which suppress tumor growth both in vitro and in vivo ( 36 , 37 ) . The effect of ATF4 deletion inducing ferroptosis suggests a treatment strategy of combining immunotherapy with ATF4 pathway inhibition. In this study, to assess the efficacy of the combinatorial therapy, xenograft tumors transplanted with RenCa cells were treated with ZZW115, anti-PD1, or both. The weight of combinatorial-treated tumors was found to be significantly decreased compared with either tumors treated with DMSO or a single agent (Fig. 7 e). Consistent with ATF4 deletion, treatment with ZZW115 remarkably promoted CD4 + and CD8 + T cell infiltration in vivo (Fig. 7 f). Collectively, our data indicate that besides impairing proliferation and motility and enhancing apoptosis and ferroptosis in tumor cells, ATF4 deletion may increase CD4 + and CD8 + T cell infiltration and promote antitumor immunity to suppress tumor growth in vivo, indicating the potential value of targeting ATF4 in treating ccRCC. 4. Discussion During tumor development, cancer cells encounter various stressful environmental conditions, such as hypoxia, nutrition deprivation, and the effect of drugs ( 38 – 41 ) . Several proteins are activated to initiate adaptive pathways in response to stress. ATF4, a stress-induced transcription factor, is usually elevated to regulate its target genes exerting corresponding functions, and this facilitates cancer cell survival, growth, development, and progression ( 10 – 12 ) . In this study, ATF4 knockdown was found to diminish ccRCC cell proliferation, migration, and invasion by suppressing mTOR activity and the EMT pathway (Figs. 2 c, 2 d, and 2 f). Our data also suggest a potentially negative role of ATF4 in ferroptosis, and ATF4 deletion triggered ferroptosis by increasing Fe 2+ accumulation in ccRCC (Figs. 6 c– 6 e). Reports have described how ATF4 promotes ER stress-induced cell death by targeting downstream genes ( 42 , 43 ) , and in some contexts, ATF4 has been found to induce a pro-apoptotic effect by dimerizing with C/EBP homologous protein (CHOP) ( 44 – 46 ) . Interestingly, in our study, ATF4 knockdown promoted apoptosis in ccRCC cells (Figs. 2 e and 2 f), which suggests that ATF4 plays an anti-apoptotic function in a CHOP-independent manner. It was hypothesized that ATF4 deletion could increase vulnerability to cellular stress during tumor growth, thereby resulting in the indirect induction of cell death. Consistently, inhibiting cell growth by ATF4 deletion was partially rescued by ferrostatin-1 treatment (Fig. 6 g). The transcriptional regulation of ATF4 is, therefore, complex, and multiple pathways may be involved in ATF4-mediated cell biological processes. ATF4 exhibits a dual functionality within tumor cells ( 47 ) . On the one hand, it regulates genes associated with amino acid transportation and metabolism, protection against oxidative stress, and maintenance of protein homeostasis ( 48 , 49 ) . On the other hand, ATF4 can trigger apoptosis, cell-cycle arrest, and senescence ( 50 – 56 ) . ATF4 induces GADD34, leading to a negative feedback loop involving dephosphorylation of eIF2a and restoration of translation ( 49 ) . ATF4 exerts a dual effect on mTOR activity, whereby it promotes amino acid availability through autophagy and concurrently upregulates adaptive genes. Conversely, ATF4 also induces the expression of mTOR repressors, namely, SESN2, DDIT4, and REDD1 ( 57 – 59 ) . The ATF4-initiated transcriptional program concurrently diminishes the expression of the anti-apoptotic BCL2 protein while augmenting pro-apoptotic signaling mediated by the proteins BIM, NOXA, and PUMA ( 60 , 61 ) . Consequently, the outcome of ATF4 activation is highly context-dependent. Based on previous studies and the results obtained in this study, we conclude that NUPR1 is a potential key target gene of ATF4 and serves as a predictor of ccRCC ( 62 ) . ATF4 and NUPR1 were upregulated in ccRCC tissues and significantly associated with poor outcomes (Figs. 1 c, 1 f, 4 a, 4 e, and 4 f), and the NUPR1 inhibitor, ZZW115, suppressed ccRCC tumor growth in vitro and in vivo (Figs. 5 a and 7 e). In addition to the regulation of apoptosis, it was identified that ATF4 is required for NUPR1 expression in response to ferroptosis activators in ccRCC. In some cases, acquired sensitivity to ferroptosis has been attributed to the specific metabolic reprogramming of cancer cells, and this provides new opportunities for treating tumors that are insensitive to regular therapy. Currently, triggering ferroptosis is a promising approach for inducing tumor cell death, especially in tumors resistant to traditional treatment strategies ( 63 ) . NUPR1, a stress-induced protein, engages in a positive feedback loop with ATF4. NUPR1 was found to present nuclear localization complexed to other protein(s) or nucleic acids in sub-confluent cells but localized throughout the whole cell in those grown to high density or an arrested Go state of the cells, indicating the nuclear import and export of NUPR1. It is reported that NUPR1 exists in the cell as part of protein complexes and directs their subcellular localization to regulate the protein function ( 64 ) . An architectural role in transcription, already proposed for NUPR1, is compatible with nuclear localization ( 65 ) . ATF4 is confirmed to regulate NUPR1 transcriptionally ( 66 ) . As our results confirm, NUPR1 and ATF4 present a nuclear localization, suggesting the function of transcription. However, a cytoplasmic localization of NUPR1 is reported in papillary carcinomas, which might reflect disease progression. Further investigations must be conducted to uncover the molecular mechanism of difference in the localization of NUPR1 based on cell density, environmental changes, tumor type, and other factors. Nevertheless, our data support that ATF4/NUPR1 axis plays a pro-survival role by activating the AKT/mTOR pathway and decreasing the apoptosis and ferroptosis cell death in ccRCC. Furthermore, the current study provides evidence that targeting ATF4/NUPR1 with or without a ferroptosis inducer may be a new effective treatment for ccRCC. Ferroptotic cancer cells can release a set of immunostimulatory signals, allowing immune cells to properly locate the site of dying tumor cells ( 63 ) . Several studies have demonstrated that ferroptosis inducers could be associated with TME features and enhance the efficacy of immune checkpoint inhibitor immunotherapy ( 17 , 35 , 36 , 67 ) . Immunostimulatory signals released by ferroptotic cancer cells can promote the phenotypic maturation of dendritic cells and increase the efficiency of macrophages, particularly M1-like tumor-associated macrophages, in the phagocytosis of ferroptotic cancer cells, strengthening CD8 + T cell-mediated tumor suppression. Interferon-γ secreted by CD8 + T cells represses solute carrier family 7 member 11 (SLC7A11) expression in cancer cells, which promotes cancer cell ferroptosis. In addition, the induction of ferroptosis can impair regulatory T (Treg) cells, myeloid-derived suppressor cells (MDSCs), and M2-like TAMs, augmenting antitumor immunity ( 68 ) . Memory CD4 + T cells and M0 macrophages can be activated via ferroptosis-related lncRNA in hepatocellular carcinoma ( 69 ) . Our study consistently confirmed, in vivo, that activated CD8 + T cells enhanced ferroptosis-specific lipid peroxidation in tumor cells. In turn, increased ferroptosis contributes to the antitumor efficacy of immunotherapy. Therefore, targeting the tumor ferroptosis pathway would be a therapeutic approach to use in combination with checkpoint blockade ( 36 ) . The present study confirms that ATF4 deletion and NUPR1 inhibitor treatment increased CD4 + and CD8 + T cell infiltration, which is presumably a result of ferroptosis. However, additional studies are required to identify these mechanisms experimentally. Therapeutic interventions can also contribute to a stressed state ( 70 ) . Unsurprisingly, ATF4 mediates resistance to therapeutic agents in various cancers. NUPR1 expression is consistently associated with resistance to several drugs ( 71 ) . For example, NUPR1 silencing reverses sorafenib resistance in ccRCC by targeting the PTEN/AKT/mTOR pathway ( 62 ) . Therefore, suppressing the ATF4/NUPR1 pathway may reduce the capacity for drug-induced stress in tumor cells, resulting in cell death induction, proliferation inhibition, and increased therapeutic efficacy. The potential beneficial effects of NUPR1 inhibitors in combination with anti-PD1 in ccRCC were confirmed via animal experiments. 5. Conclusions ATF4 knockdown inhibits proliferation and motility and induces apoptosis and ferroptosis via NUPR1 in ccRCC, increasing the infiltration of CD4 + and CD8 + T cells and enhancing antitumor immunity. Therefore, targeting ATF4/NUPR1 may be a potential alternative therapeutic strategy for controlling ccRCC progression. Further studies are required to elucidate the mechanism whereby ATF4 exerts antitumor immunity. Declarations Funding Not applicable. Conflicts of interest The authors declare no conflicts of interest. Data Availability Statement The data that support the findings of this study are available in the methods and/or supplementary material of this article. Code availability Not applicable. Authors' contributions Xin Ma, Yan Huang, Hongzhao Li and Yongliang Lu conceived the project and designed the experiments. Weihao Chen and Yundong Xuan performed the cell experiments.Yongliang Lu, Yundong Xuan, Shengpan Wu and Hanfeng Wang conducted the plasmid construction and molecular experiments. Tao Guo, Chenfeng Wang and Shuo Tian provided the human tissue samples and clinical information. Yongliang Lu and Yundong Xuan performed the immunofluorescence assays. Yongliang Lu, Weihao Chen, Shengpan Wu, Xupeng zhao, Wenlei Zhao and Tao Guo performed the IHC and H&E staining. Yongliang Lu, Weihao Chen, Yundong Xuan, Shengpan Wu, Huaikang Li, Dong Lai and Tao Guo performed the animal experiments. Yongliang Lu, Xiubin Li and Xing Huang performed the bioinformatics analyses and conducted the RNA sequencing analysis. Yongliang Lu Weihao chen and Yan Huang analyzed and interpreted the data. Yan Huang, Hongzhao Li and Xin Ma supervised the overall execution of the experiments. The manuscript was written by Yongliang Lu with support from Baojun Wang, Xu Zhang, Yan Huang, Hongzhao Li and Xin Ma. All authors have read and approved the final manuscript. Acknowledgments Not applicable Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Research Ethics Committee of the PLA General Hospital. The animal study protocol was approved by the Institutional Animal Care and Use Committee of the PLA General Hospital. The study is reported in accordance with ARRIVE guidelines. Consent to publish Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patients to publish this paper. References Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Piñeros M, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144(8):1941–53. Siegel RL, Miller KD, Fuchs HE, Jemal A, Cancer Statistics. 2021. CA Cancer J Clin. 2021;71(1):7–33. Cohen HT, McGovern FJ. Renal-cell carcinoma. N Engl J Med. 2005;353(23):2477–90. Ricketts CJ, Crooks DR, Sourbier C, Schmidt LS, Srinivasan R, Linehan WM. SnapShot: Renal Cell Carcinoma. Cancer Cell. 2016;29(4):610–e1. Ljungberg B, Albiges L, Abu-Ghanem Y, Bedke J, Capitanio U, Dabestani S et al. European Association of Urology Guidelines on Renal Cell Carcinoma: The 2022 Update. Eur Urol. 2022. Dejeans N, Barroso K, Fernandez-Zapico ME, Samali A, Chevet E. Novel roles of the unfolded protein response in the control of tumor development and aggressiveness. Semin Cancer Biol. 2015;33:67–73. Maurel M, McGrath EP, Mnich K, Healy S, Chevet E, Samali A. Controlling the unfolded protein response-mediated life and death decisions in cancer. Semin Cancer Biol. 2015;33:57–66. Urra H, Dufey E, Avril T, Chevet E, Hetz C. Endoplasmic Reticulum Stress and the Hallmarks of Cancer. Trends Cancer. 2016;2(5):252–62. Sullivan LB, Chandel NS. Mitochondrial reactive oxygen species and cancer. Cancer Metab. 2014;2:17. Kilberg MS, Shan J, Su N. ATF4-dependent transcription mediates signaling of amino acid limitation. Trends Endocrinol Metab. 2009;20(9):436–43. Luhr M, Torgersen ML, Szalai P, Hashim A, Brech A, Staerk J, et al. The kinase PERK and the transcription factor ATF4 play distinct and essential roles in autophagy resulting from tunicamycin-induced ER stress. J Biol Chem. 2019;294(20):8197–217. Rozpedek W, Pytel D, Mucha B, Leszczynska H, Diehl JA, Majsterek I. The Role of the PERK/eIF2α/ATF4/CHOP Signaling Pathway in Tumor Progression During Endoplasmic Reticulum Stress. Curr Mol Med. 2016;16(6):533–44. Liu M, Wang L, Xia X, Wu Y, Zhu C, Duan M, et al. Regulated lytic cell death in breast cancer. Cell Biol Int. 2022;46(1):12–33. Liang C, Zhang X, Yang M, Dong X. Recent Progress in Ferroptosis Inducers for Cancer Therapy. Adv Mater. 2019;31(51):e1904197. Lee AS. Glucose-regulated proteins in cancer: molecular mechanisms and therapeutic potential. Nat Rev Cancer. 2014;14(4):263–76. Sun X, Ou Z, Xie M, Kang R, Fan Y, Niu X, et al. HSPB1 as a novel regulator of ferroptotic cancer cell death. Oncogene. 2015;34(45):5617–25. Bai D, Feng H, Yang J, Yin A, Lin X, Qian A, et al. Genomic analysis uncovers prognostic and immunogenic characteristics of ferroptosis for clear cell renal cell carcinoma. Mol Ther Nucleic Acids. 2021;25:186–97. Gao Y, Li H, Ma X, Fan Y, Ni D, Zhang Y, et al. KLF6 Suppresses Metastasis of Clear Cell Renal Cell Carcinoma via Transcriptional Repression of E2F1. Cancer Res. 2017;77(2):330–42. Hamanishi J, Mandai M, Iwasaki M, Okazaki T, Tanaka Y, Yamaguchi K, et al. Programmed cell death 1 ligand 1 and tumor-infiltrating CD8 + T lymphocytes are prognostic factors of human ovarian cancer. Proc Natl Acad Sci U S A. 2007;104(9):3360–5. Horikawa N, Abiko K, Matsumura N, Hamanishi J, Baba T, Yamaguchi K, et al. Expression of Vascular Endothelial Growth Factor in Ovarian Cancer Inhibits Tumor Immunity through the Accumulation of Myeloid-Derived Suppressor Cells. Clin Cancer Res. 2017;23(2):587–99. Rao X, Huang X, Zhou Z, Lin X. An improvement of the 2ˆ(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath. 2013;3(3):71–85. Sandi MJ, Hamidi T, Malicet C, Cano C, Loncle C, Pierres A, et al. p8 expression controls pancreatic cancer cell migration, invasion, adhesion, and tumorigenesis. J Cell Physiol. 2011;226(12):3442–51. Emma MR, Iovanna JL, Bachvarov D, Puleio R, Loria GR, Augello G, et al. NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance. Cell Death Dis. 2016;7(6):e2269. Guo X, Wang W, Hu J, Feng K, Pan Y, Zhang L, et al. Lentivirus-mediated RNAi knockdown of NUPR1 inhibits human nonsmall cell lung cancer growth in vitro and in vivo. Anat Rec (Hoboken). 2012;295(12):2114–21. Kim KS, Jin DI, Yoon S, Baek SY, Kim BS, Oh SO. Expression and roles of NUPR1 in cholangiocarcinoma cells. Anat Cell Biol. 2012;45(1):17–25. Li J, Ren S, Liu Y, Lian Z, Dong B, Yao Y, et al. Knockdown of NUPR1 inhibits the proliferation of glioblastoma cells via ERK1/2, p38 MAPK and caspase-3. J Neurooncol. 2017;132(1):15–26. Zeng C, Li X, Li A, Yi B, Peng X, Huang X, et al. Knockdown of NUPR1 inhibits the growth of U266 and RPMI8226 multiple myeloma cell lines via activating PTEN and caspase activation–dependent apoptosis. Oncol Rep. 2018;40(3):1487–94. Zhou C, Xu J, Lin J, Lin R, Chen K, Kong J, et al. Long Noncoding RNA FEZF1-AS1 Promotes Osteosarcoma Progression by Regulating the miR-4443/NUPR1 Axis. Oncol Res. 2018;26(9):1335–43. Yu J, Zhu H, Li R, Jiang Q, Luan W, Shi J, et al. Oncogenic Role of NUPR1 in Ovarian Cancer. Onco Targets Ther. 2020;13:12289–300. Jiang L, Wang W, Li Z, Zhao Y, Qin Z. NUPR1 participates in YAP-mediate gastric cancer malignancy and drug resistance via AKT and p21 activation. J Pharm Pharmacol. 2021;73(6):740–8. Tang K, Zhang Z, Bai Z, Ma X, Guo W, Wang Y. Enhancement of gemcitabine sensitivity in pancreatic cancer by co-regulation of dCK and p8 expression. Oncol Rep. 2011;25(4):963–70. Jiang W, Zhang C, Zhang X, Sun L, Li J, Zuo J. CircRNA HIPK3 promotes the progression of oral squamous cell carcinoma through upregulation of the NUPR1/PI3K/AKT pathway by sponging miR-637. Ann Transl Med. 2021;9(10):860. Liu J, Song X, Kuang F, Zhang Q, Xie Y, Kang R, et al. NUPR1 is a critical repressor of ferroptosis. Nat Commun. 2021;12(1):647. Lan W, Santofimia-Castaño P, Swayden M, Xia Y, Zhou Z, Audebert S et al. ZZW-115-dependent inhibition of NUPR1 nuclear translocation sensitizes cancer cells to genotoxic agents. JCI Insight. 2020;5(18). Wang S, Chen S, Ying Y, Ma X, Shen H, Li J, et al. Comprehensive Analysis of Ferroptosis Regulators With Regard to PD-L1 and Immune Infiltration in Clear Cell Renal Cell Carcinoma. Front Cell Dev Biol. 2021;9:676142. Wang W, Green M, Choi JE, Gijón M, Kennedy PD, Johnson JK, et al. CD8(+) T cells regulate tumour ferroptosis during cancer immunotherapy. Nature. 2019;569(7755):270–4. Tang R, Xu J, Zhang B, Liu J, Liang C, Hua J, et al. Ferroptosis, necroptosis, and pyroptosis in anticancer immunity. J Hematol Oncol. 2020;13(1):110. Garris CS, Pittet MJ. ER Stress in Dendritic Cells Promotes Cancer. Cell. 2015;161(7):1492–3. Todoric J, Antonucci L, Di Caro G, Li N, Wu X, Lytle NK, et al. Stress-Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas. Cancer Cell. 2017;32(6):824–39e8. Linares JF, Cordes T, Duran A, Reina-Campos M, Valencia T, Ahn CS, et al. ATF4-Induced Metabolic Reprograming Is a Synthetic Vulnerability of the p62-Deficient Tumor Stroma. Cell Metab. 2017;26(6):817–29e6. Seton-Rogers S, Oncogenes. Coping with stress. Nat Rev Cancer. 2017;17(2):76–7. Armstrong JL, Flockhart R, Veal GJ, Lovat PE, Redfern CP. Regulation of endoplasmic reticulum stress-induced cell death by ATF4 in neuroectodermal tumor cells. J Biol Chem. 2010;285(9):6091–100. Huang H, Hua X, Liu N, Li X, Liu S, Chen X, et al. Anacardic acid induces cell apoptosis associated with induction of ATF4-dependent endoplasmic reticulum stress. Toxicol Lett. 2014;228(3):170–8. Han J, Back SH, Hur J, Lin YH, Gildersleeve R, Shan J, et al. ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat Cell Biol. 2013;15(5):481–90. Matsumoto H, Miyazaki S, Matsuyama S, Takeda M, Kawano M, Nakagawa H, et al. Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression. Biol Open. 2013;2(10):1084–90. Teske BF, Fusakio ME, Zhou D, Shan J, McClintick JN, Kilberg MS, et al. CHOP induces activating transcription factor 5 (ATF5) to trigger apoptosis in response to perturbations in protein homeostasis. Mol Biol Cell. 2013;24(15):2477–90. Wortel IMN, van der Meer LT, Kilberg MS, van Leeuwen FN. Surviving Stress: Modulation of ATF4-Mediated Stress Responses in Normal and Malignant Cells. Trends Endocrinol Metab. 2017;28(11):794–806. Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, et al. An Integrated Stress Response Regulates Amino Acid Metabolism and Resistance to Oxidative Stress. Mol Cell. 2003;11(3):619–33. Novoa I, Zhang Y, Zeng H, Jungreis R, Harding HP, Ron D. Stress-induced gene expression requires programmed recovery from translational repression. Embo j. 2003;22(5):1180–7. Frank CL, Ge X, Xie Z, Zhou Y, Tsai LH. Control of activating transcription factor 4 (ATF4) persistence by multisite phosphorylation impacts cell cycle progression and neurogenesis. J Biol Chem. 2010;285(43):33324–37. Han J, Back SH, Hur J, Lin Y-H, Gildersleeve R, Shan J, et al. ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat Cell Biol. 2013;15(5):481–90. Lin H-K, Chen Z, Wang G, Nardella C, Lee S-W, Chan C-H, et al. Skp2 targeting suppresses tumorigenesis by Arf-p53-independent cellular senescence. Nature. 2010;464(7287):374–9. Hiramatsu N, Messah C, Han J, Lavail M, Lin J. Translational and post-translational regulation of XIAP by eIF2α and ATF4 promotes ER stress๊nduced cell death during the unfolded protein response (151.5). FASEB J. 2014;28:1. Matsumoto H, Miyazaki S, Matsuyama S, Takeda M, Kawano M, Nakagawa H, et al. Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression. Biology Open. 2013;2(10):1084–90. Ohoka N, Yoshii S, Hattori T, Onozaki K, Hayashi H. TRB3, a novel ER stress-inducible gene, is induced via ATF4–CHOP pathway and is involved in cell death. EMBO J. 2005;24(6):1243–55. Ishizawa J, Kojima K, Chachad D, Ruvolo P, Ruvolo V, Jacamo RO, et al. ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies. Sci Signal. 2016;9(415):ra17. Guan B-J, Krokowski D, Majumder M, Schmotzer CL, Kimball SR, Merrick WC, et al. Translational Control during Endoplasmic Reticulum Stress beyond Phosphorylation of the Translation Initiation Factor eIF2α*. J Biol Chem. 2014;289(18):12593–611. Yu X, Long YC. Autophagy modulates amino acid signaling network in myotubes: differential effects on mTORC1 pathway and the integrated stress response. FASEB J. 2015;29(2):394–407. Dennis MD, McGhee NK, Jefferson LS, Kimball SR. Regulated in DNA damage and development 1 (REDD1) promotes cell survival during serum deprivation by sustaining repression of signaling through the mechanistic target of rapamycin in complex 1 (mTORC1). Cell Signal. 2013;25(12):2709–16. Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol. 2012;13(2):89–102. Szegezdi E, Logue SE, Gorman AM, Samali A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006;7(9):880–5. He W, Cheng F, Zheng B, Wang J, Zhao G, Yao Z, et al. NUPR1 is a novel potential biomarker and confers resistance to sorafenib in clear cell renal cell carcinoma by increasing stemness and targeting the PTEN/AKT/mTOR pathway. Aging. 2021;13(10):14015–38. Friedmann Angeli JP, Krysko DV, Conrad M. Ferroptosis at the crossroads of cancer-acquired drug resistance and immune evasion. Nat Rev Cancer. 2019;19(7):405–14. Valacco MP, Varone C, Malicet C, Cánepa E, Iovanna JL, Moreno S. Cell growth-dependent subcellular localization of p8. J Cell Biochem. 2006;97(5):1066–79. Hoffmeister A, Ropolo A, Vasseur S, Mallo GV, Bodeker H, Ritz-Laser B, et al. The HMG-I/Y-related protein p8 binds to p300 and Pax2 trans-activation domain-interacting protein to regulate the trans-activation activity of the Pax2A and Pax2B transcription factors on the glucagon gene promoter. J Biol Chem. 2002;277(25):22314–9. Jin HO, Seo SK, Woo SH, Choe TB, Hong SI, Kim JI, et al. Nuclear protein 1 induced by ATF4 in response to various stressors acts as a positive regulator on the transcriptional activation of ATF4. IUBMB Life. 2009;61(12):1153–8. Yu B, Choi B, Li W, Kim DH. Magnetic field boosted ferroptosis-like cell death and responsive MRI using hybrid vesicles for cancer immunotherapy. Nat Commun. 2020;11(1):3637. Lei G, Zhuang L, Gan B. Targeting ferroptosis as a vulnerability in cancer. Nat Rev Cancer. 2022;22(7):381–96. Xu Z, Peng B, Liang Q, Chen X, Cai Y, Zeng S, et al. Construction of a Ferroptosis-Related Nine-lncRNA Signature for Predicting Prognosis and Immune Response in Hepatocellular Carcinoma. Front Immunol. 2021;12:719175. Nagelkerke A, Bussink J, Sweep FC, Span PN. The unfolded protein response as a target for cancer therapy. Biochim Biophys Acta. 2014;1846(2):277–84. Malicet C, Giroux V, Vasseur S, Dagorn JC, Neira JL, Iovanna JL. Regulation of apoptosis by the p8/prothymosin alpha complex. Proc Natl Acad Sci U S A. 2006;103(8):2671–6. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.rar graphicalabstract.png The graphical abstract was drawn using Figdraw online (free website: www.figdraw.com) Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 14 Aug, 2024 Reviews received at journal 13 Aug, 2024 Reviewers agreed at journal 09 Aug, 2024 Reviews received at journal 17 Jul, 2024 Reviewers agreed at journal 12 Jul, 2024 Reviewers invited by journal 28 Mar, 2024 Editor assigned by journal 16 Feb, 2024 Submission checks completed at journal 16 Feb, 2024 First submitted to journal 16 Jan, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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(b) Kaplan–Meier analysis of OS for all ccRCC patients with low or high ATF4 expression levels in the TCGA dataset. (c) Western blot experiment was performed to evaluate the protein level of ATF4 in five paired ccRCC tissue samples (“T” means tumor tissue, and “N” means paired normal tissue). (d) Representative images of ATF4 IHC staining with high or low expression level in ccRCC tumor tissues by TMAs. (e) IHC representative images and analysis of ATF4 expression in tumor tissues (n = 199) and normal tissues (n = 143) by TMAs. (f) Kaplan–Meier analysis of ccRCC over survival based on ATF4 expression level in ccRCC TMAs. The p-value was calculated using the log-rank test. (g) Comparison of ATF4 IHC scores among groups with different tumor TNM stages and tumor grades. The p-values were calculated using the Mann–Whitney U test. (h) Association of ATF4 expression with clinical and pathological characteristics of 199 ccRCC patients in TMAs. The data are presented in terms of means ± SEM. The p-values were estimated using the Mann–Whitney U test (*p \u0026lt; .05, ** p \u0026lt; .01, ***p \u0026lt; .001, ****p \u0026lt; .0001)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3870049/v1/a43c9a7c64b9d50da317bf06.png"},{"id":51388673,"identity":"30e5977c-5937-412a-9c47-9d12ea6a4f2e","added_by":"auto","created_at":"2024-02-20 18:07:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1437183,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATF4 knockdown attenuates ccRCC cell proliferation, migration, and invasion and induces apoptosis \u0026nbsp;\u003c/strong\u003e(a) Relative ATF4 protein levels in HEK293T, HKC cells, and a panel of ccRCC cell lines. (b) ATF4 expression levels in 786-O and SN12 cells infected with shATF4 or NC for four days were detected via Western blot. (c) The indicated cells are infected with shATF4 for four days, followed by bright-field imaging (left) and quantification (right). Optical density (OD) values at 450 nm were used to compare cell growth ability between different groups. (d) Transwell assay indicates impaired abilities of migration and invasion of 786-O and SN12 cells treated with shATF4. Representative graphs (left). Bar graph showing the statistical results (right). (e) Effect of ATF4 knockdown on the apoptosis rate in 786-O and SN12 cells. (f) Western blot analysis of mTOR, EMT, and apoptosis-related gene expression in 786-O cells with NC and shATF4. (g) BALB/c nude mice underwent orthotopic implantation of SN12 cells, stably expressing control shRNA or shATF4, and then tumor masses were resected on day 21. Tumor weights were expressed in terms of mean ± SEM, n = 6 for each group. (h) H\u0026amp;E and IHC analyses of tissues harvested from the mice tumor masses stained for ATF4, ki67, and cleaved caspase-3. All functional assays above were independently repeated thrice in triplicate. The data are presented herein as mean ± SEM. p-values were calculated using the two-tailed Student’s t-test (*p \u0026lt; .05, ** p \u0026lt; .01, ***p \u0026lt; .001, ****p \u0026lt; .0001)\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3870049/v1/d6e43c7ba1e51d90a0219f2b.png"},{"id":51388703,"identity":"0af791fb-8259-4995-9e94-149ac2191ca1","added_by":"auto","created_at":"2024-02-20 18:07:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":901590,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATF4 deficiency downregulates NUPR1 expression in ccRCC \u003c/strong\u003e(a) Heatmap analysis of DEG function clustering in the ccRCC from NC and shATF4 groups (b) Kyoto KEGG pathway enrichment and GO enrichment analysis among DEGs in 786-O cells. (c) Venn diagram illustrating overlap among DEGs and target gene lists. DEGs 1 (NC vs. shATF4-1), DEGs 2 (NC vs. shATF4-2), target genes (the predicted gene list transcription-regulated by ATF4). (d) Volcano plot depicting the gene expression differences between NC and shATF4 groups. The horizontal axis represents the log-ratio (gene expression fold change in different samples), while the vertical axis represents the probability for each gene of being differentially expressed. The 75 genes in (c) are shown. (e) NUPR1 was verified via qRT-PCR experiment in ATF4 knockdown 786-O and SN12 cells. Data are presented in terms of mean ± SEM, n = 3. The p-values were calculated using the two-tailed Student’s t-test (*p \u0026lt; .05, ** p \u0026lt; .01, ***p \u0026lt; .001, ****p \u0026lt; .0001). (f) NUPR1 expression levels were detected by Western blot in ATF4 knockdown 786-O and SN12 cells. (g) Expression correlation of ATF4 and NUPR1 based on the IHC score in ccRCC tissue by TMAs (n = 199), representative IHC staining images in two ccRCC patients (left), the statistical result of correlation analysis (right)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3870049/v1/dcc8ebf10f7cff9fccd85335.png"},{"id":51388701,"identity":"fcf82caa-0f45-4c3a-8fa5-1a0c4cbb8e0e","added_by":"auto","created_at":"2024-02-20 18:07:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":920556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNUPR1 expression correlates with the progression and prognosis of ccRCC patients\u003c/strong\u003e(a) The mRNA levels of NUPR1 at different clinical stages of ccRCC tissues in the TCGA dataset. (b) Kaplan–Meier analysis of OS for all ccRCC patients with low or high NUPR1 expression levels in the TCGA dataset. (c) Representative images of NUPR1 IHC staining with high or low expression levels in ccRCC tumor tissues by TMAs. (d) IHC representative images and analysis of NUPR1 expression in tumor tissues (n = 199) and normal tissues (n = 143) by TMAs. (e) The NUPR1 expression in different clinical TNM stages and histological grades of ccRCC patients (n = 199). (f) Kaplan–Meier analysis of OS for 199 ccRCC patients in TMAs with low or high NUPR1 expression levels. The data are represented as means ± SEM. The p-values were calculated using the two-tailed Student’s t-test, Mann–Whitney U test, or log-rank test (*p \u0026lt; .05, ** p \u0026lt; .01, ***p \u0026lt; .001, ****p \u0026lt; .0001)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3870049/v1/5e24091692478a05db1f1249.png"},{"id":51388674,"identity":"888ee056-f0ce-4f92-a238-d67ec64ffa25","added_by":"auto","created_at":"2024-02-20 18:07:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1246147,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZZW115 inhibits cell growth by inducing apoptosis and ferroptosis \u003c/strong\u003e(a) Cell proliferation assay was performed in ccRCC cells (786-O and SN12) treated with ZZW115 for 24 h in a dose-dependent manner. Cell death was assayed using a CCK8 kit. (b) Transwell assay indicated impaired abilities of migration and invasion of 786-O and SN12 cells treated with ZZW115 (10 μM) for 24 h. Representative graphs (left). Bar graph showing the statistical results (right). (c-d) Representative images of TUNEL immunofluorescent staining and flow cytometry analyses for apoptosis of 786-O and SN12 cells treated with DMSO or ZZW115 (10 μM) for 24 h. (e) Western blot of EMT, apoptosis-related, and AKT/mTOR genes expression in 786-O cells with DMSO or ZZW115 (10 μM) for 24 h. (f) Levels of lipid ROS were analyzed in 786-O cells with DMSO or ZZW115 (10 μM) for 24 h. The data are presented as mean ± SEM，n = 3. The p-values were calculated using the two-tailed Student’s t-test (*p \u0026lt; .05, ** p \u0026lt; .01, ***p \u0026lt; .001, ****p \u0026lt; .0001)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3870049/v1/94cb940959f1b06f031b3614.png"},{"id":51388706,"identity":"5559bdc3-963d-4837-af99-97638b40084f","added_by":"auto","created_at":"2024-02-20 18:07:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":858615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of ATF4 induces ferroptosis and inhibits cell proliferation by targeting NUPR1\u003c/strong\u003e (a) Western blot indicates ATF4 protein expression in 786-O cells following treatment with erastin (0–5 μM) for 24 h. (b) ATF4 knockdown inhibited erastin-induced (2.5 μM, 24 h) NUPR1 protein expression. (c) Scanning electron microscopy of 786-O cells transfected with NC or shATF4. Mitochondrion (arrow). (d-e) Representative immunofluorescent microscope images and flow cytometry analyses of lipid peroxidation levels reported by BODIPY-C11 in 786-O cells transfected with NC or shATF4 in the absence or presence of ferrostatin-1. (f) Representative immunofluorescent microscope images of FerroOrange (left) and statistical result (right) for relative Fe\u003csup\u003e2+ \u003c/sup\u003elevel\u003csup\u003e \u003c/sup\u003eof 786-O cells transfected with NC or shATF4. (g) 786-O cells were infected with shATF4 for four days in the absence or presence of ferrostatin-1, followed by bright-field imaging (left) and quantification (right). OD values at 450 nm were used to compare cell growth ability between different groups. The data are presented as mean ± SEM，n = 3. The p-values were estimated using the two-tailed Student’s t-test (*p \u0026lt; .05, ** p \u0026lt; .01, ***p \u0026lt; .001, ****p \u0026lt; .0001)\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3870049/v1/5da12c257e66949d644b182b.png"},{"id":51388705,"identity":"2b1f7764-37dc-4355-bc67-9993840e4287","added_by":"auto","created_at":"2024-02-20 18:07:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1586698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eATF4 depletion remodels TME and promotes antitumor immunity\u003c/strong\u003e (a) BALB/c mice underwent orthotopic implantation of RenCa cells, stably expressing control shRNA or shATF4 respectively, and then tumor masses were resected on day 14. N = 4 for each group. (b) Representative H\u0026amp;E staining images and quantitative analysis for the amount of lungs with metastatic nodules (black arrow indicates metastatic nodules). (c) IHC analysis of tissues harvested from the mice tumor masses stained for NUPR1, CD8, CD4, and granzyme B. (d) Representative images and statistical results of CD8 and CD4 IHC staining with ATF4 high or low expression level in ccRCC tumor tissues by TMAs (n = 199). (e) Tumor growth of RenCa cells with ZZW115 and/or anti-PD1 therapy treated in BALB/c mice. n = 5for each group. (f) IHC analysis of tumor tissue harvested from the mice treated with DMSO or ZZW115 stained for CD8 and CD4. The data are represented in terms of mean ± SEM, and p-values were calculated using the Mann–Whitney U test (*p \u0026lt; .05, ** p \u0026lt; .01, ***p \u0026lt; .001, ****p \u0026lt; .0001)\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-3870049/v1/89102c48de65efef31af8f98.png"},{"id":51388790,"identity":"3a8a8fc6-ab94-40b9-8f55-c0de3bd48da0","added_by":"auto","created_at":"2024-02-20 18:07:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4284423,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3870049/v1/5f6df021-75ea-4793-982e-f316eab53051.pdf"},{"id":51388704,"identity":"91f7deb0-6206-42a0-9768-40c6bbb5c892","added_by":"auto","created_at":"2024-02-20 18:07:19","extension":"rar","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3251088,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.rar","url":"https://assets-eu.researchsquare.com/files/rs-3870049/v1/d11d3f58fbca5821897d61af.rar"},{"id":51388676,"identity":"9499fe89-b4a7-4946-8282-4f743da08c76","added_by":"auto","created_at":"2024-02-20 18:07:18","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":473962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eThe graphical abstract was drawn using Figdraw online (free website: www.figdraw.com)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-3870049/v1/6764a9b34663602c8b32bf7d.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"ATF4/NUPR1 axis promotes cancer cell survival and mediates immunosuppression in clear cell renal cell carcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRenal cell carcinoma (RCC) is the sixth leading cause of cancer-related deaths globally, with a slight increase in associated morbidity and mortality in recent years\u003csup\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/sup\u003e. Approximately 30% of RCC patients are initially diagnosed with advanced or metastatic disease due to occult early symptoms and a lack of markers\u003csup\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/sup\u003e, and approximately 75\u0026ndash;80% of the pathological RCC types are clear cell renal cell carcinoma (ccRCC)\u003csup\u003e(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/sup\u003e. Although surgery is potentially curative for ccRCC when detected at an early stage, postoperative recurrence remains common, and tyrosine kinase inhibitors and immunotherapy for advanced ccRCC remain unsatisfactory owing to drug resistance\u003csup\u003e(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/sup\u003e. Consequently, it is crucial to acquire in-depth insights into the molecular mechanisms underlying ccRCC occurrence and development to identify new targets and develop therapies for its treatment.\u003c/p\u003e \u003cp\u003eRapidly growing tumors with excessive proliferation escalate the demand for protein synthesis, which contributes to endoplasmic reticulum (ER) stress\u003csup\u003e(\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/sup\u003e, and cells in the tumor core suffer from a lack of oxygen and nutrients due to insufficient and defective vascularization. Consequently, sets of adaptive genes are activated in response to these challenges \u003csup\u003e(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/sup\u003e, and there is a common downstream effector protein, ATF4, that is frequently upregulated in cancer cells. It controls the expression of target genes, most of which are involved in various salvage pathways that relieve stress and promote cell survival\u003csup\u003e(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/sup\u003e. Recent studies have revealed that ATF4 is required for distinct steps to increase amino acid availability in the autophagic pathway, and it may enhance mTOR activity by upregulating adaptive genes\u003csup\u003e(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/sup\u003e. It is thus evident that ATF4 plays a pivotal role in alleviating stress during rapid tumor growth. Ferroptosis is a form of regulated cell death characterized by iron-dependent lipid peroxidation\u003csup\u003e(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/sup\u003e. Various metabolic processes, such as those associated with iron and amino acids, have been suggested to be related to ferroptosis\u003csup\u003e(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/sup\u003e. ATF4 is known to be an adaptive regulator of cancer cell responses to various stresses, including ER stress during ferroptosis by erastin\u003csup\u003e(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003c/sup\u003e. Moreover, a recent study demonstrated that ferroptosis is related to the tumor microenvironment (TME) features of ccRCC, and it may thus play a key role in the response to immunotherapy\u003csup\u003e(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHowever, the functions and regulatory mechanisms of ATF4 remain largely unknown in ccRCC. Therefore, this study aimed to investigate the role of ATF4 in ccRCC, especially its function in ferroptosis and tumor immunology.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cp\u003e \u003cem\u003eCell culture, reagents, and transfection\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe human renal proximal tubular epithelial cell lines (HEK293T and HKC), ccRCC cell lines (A498 ACHN, Caki-1, 786-O, SN12), and murine RCC cell line (RenCa) were purchased in September 2015 from the National Platform of Experimental Cell Resources for Sci-Tech (Beijing, China) and authenticated in January 2019 using a previously described method\u003csup\u003e(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/sup\u003e. All cell lines were cultured in the recommended medium supplemented with 10% fetal bovine serum (FBS; Evergreen Co. Ltd., China) and 1% penicillin\u0026ndash;streptomycin at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e under mycoplasma-free conditions. pLKO-shATF4 plasmids with short hairpin RNAs (shRNAs) targeting human and murine ATF4 were designed by Invitrogen. The target shATF4 sequences in humans and mice are enumerated in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Plasmid construction and transfection were performed using a method described previously\u003csup\u003e(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/sup\u003e. Chemical reagents, including erastin (CAS No. 571203-78-6), ferrostatin-1 (CAS No. 347174-05-4), ZZW115 (CAS No. 801991-87-7), and anti-PD1 (CAS No. 946414-94-4) were purchased from MedChem Express (Shanghai, China).\u003c/p\u003e \u003cp\u003e \u003cem\u003eImmunohistochemistry (IHC) and analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003e The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Research Ethics Committee of the PLA General Hospital. A total of 199 pairs of ccRCC and normal tissue specimens were included from previously constructed tissue microarrays (TMAs) in our laboratory. Immunostaining of the TMA and xenograft tumor tissues was performed as previously described\u003csup\u003e(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e)\u003c/sup\u003e. The primary antibodies included rabbit anti-ATF4 (Proteintech; 10835-1-AP), rabbit anti-NUPR1 (Proteintech; 15056-1-AP), rabbit anti-CD4 (Abcam; ab183685), rabbit anti-CD8 alpha (Abcam; ab217344), rabbit anti-cleaved caspase-3 (Cell Signaling Technology; #9661), mouse anti-granzyme B (Santa Cruz; sc-8022), and rabbit anti-Ki-67 (Cell Signaling Technology; #9129). TMA samples were classified according to the intensity of ATF4 or NUPR1 staining, and their expressions were scored as follows: 0, negative; 1, very weak; 2, moderate; and 3, strong. Cases with scores of 0/1 and 2/3 were defined as the ATF4 or NUPR1 low- and high-expression groups, respectively. Tumor-infiltrating immune cells were evaluated as previously described\u003csup\u003e(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cem\u003eReal-time PCR\u003c/em\u003e \u003c/p\u003e \u003cp\u003eIn accordance with the manufacturer\u0026rsquo;s protocol, total RNA was extracted from cells using the PARISTM Kit (Applied Biosystems) and then reverse-transcribed to obtain cDNA. With the Applied Biosystems 7500 Detection system, real-time PCR was performed. Relative mRNA levels were normalized to human peptidylprolyl isomerase A (PPIA) and calculated using the 2\u003csup\u003e\u0026minus;ΔΔC\u003c/sup\u003e\u003csub\u003eT\u003c/sub\u003e method\u003csup\u003e(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/sup\u003e. Supplementary Tables S2 and S3 specify the primer sequences and reaction conditions of mixtures for real-time PCR.\u003c/p\u003e \u003cp\u003e \u003cem\u003eImmunoblotting and Antibody\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWestern blot was performed as described previously\u003csup\u003e(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/sup\u003e. Primary antibodies included rabbit anti-ATF4 (Proteintech; 10835-1-AP), rabbit anti-mTOR (Cell Signaling Technology; #2983S), rabbit anti-phospho-mTOR (Ser2448) (D9C2) XP\u0026reg; (Cell Signaling Technology; #5536S), rabbit anti-S6K (Proteintech; 14485-1-AP), rabbit anti-phospho-S6K (Proteintech; 28735-1-AP), rabbit anti-4EBP1 (Cell Signaling Technology; #9644S), rabbit anti-phospho-4EBP1 (Cell Signaling Technology; #2855S), rabbit anti-N-cadherin (Proteintech; 22018-1-AP), rabbit anti-E-cadherin ((Proteintech; 20874-1-AP), rabbit anti-vimentin (Proteintech; 10366-1-AP), rabbit anti-cleaved caspase-3 (Cell Signaling Technology; #9661), rabbit-anti-BCL2 (Cell Signaling Technology; #3498S), rabbit anti-AKT (Cell Signaling Technology; #4685S), rabbit anti-phospho-Akt (Ser473) (D9E) XP\u0026reg; (Cell Signaling Technology; #4060S), and rabbit anti-NUPR1 (Proteintech; 15056-1-AP).\u003c/p\u003e \u003cp\u003e \u003cem\u003eFlow cytometry for cell apoptosis analysis and lipid ROS analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eAn Annexin V-FITC/PI kit (Abbkine Scientific Co., Ltd, KTA0002) was used to investigate the apoptotic effect of ATF4 deletion and the NUPR1 inhibitor. Lipid ROS were stained with C11-BODIPY 581/591 (RM02821, ABclonal Technology Co., Ltd, Wuhan, China) and analyzed using flow cytometry and fluorescence microscopy in accordance with the manufacturer\u0026rsquo;s protocols. The mean fluorescence intensity was estimated using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). All samples were analyzed in triplicate, and the data were analyzed using FlowJo version 10 software. Fluorescence imaging was performed using TissueFAXS (TissueGnostics GmbH, Vienna, Austria) with a Zeiss Axio Imager Z2 microscope system.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCell immunofluorescence: Terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) and Fe\u003c/em\u003e \u003csup\u003e \u003cem\u003e2+\u003c/em\u003e \u003c/sup\u003e \u003cem\u003elevel assays\u003c/em\u003e\u003c/p\u003e \u003cp\u003eCells were placed in culture dishes overnight for adhesion, and the relevant treatment was then applied. The TUNEL Apoptosis Detection Kit (Green Fluorescence; Abbkine Scientific Co., Ltd, KTA2010) or FerroOrange (Dojindo Laboratories, Beijing, China) was used in accordance with the manufacturer\u0026rsquo;s instructions to determine cellular apoptosis or Fe\u003csup\u003e2+\u003c/sup\u003e levels. Fluorescence images were obtained using TissueFAXS (TissueGnostics GmbH, Vienna, Austria). A quantitative analysis of the mean fluorescence signal intensity was conducted using the ImageJ software. All samples were analyzed in triplicate.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnimal modeling and treatment\u003c/em\u003e \u003c/p\u003e \u003cp\u003e The animal study protocol was approved by the Institutional Animal Care and Use Committee of the PLA General Hospital. Tumor cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e) with PBS or Matrigel (Corning) were implanted into the kidneys of male BALB/c (nude) mice (Vital River, Beijing, aged 4\u0026ndash;5 weeks) for the xenograft model or syngeneic graft. After three weeks, the mice were sacrificed, and bioluminescent imaging was performed. For the anti-PD1 treatment, the mice received 200 \u0026micro;g of anti-PD1 via intraperitoneal injection on days 8, 11, 14, 17, and 20, and for the NUPR1 inhibitor ZZW115 treatment, mice received 1 mg/kg ZZW115 via intraperitoneal injection daily (treatment on days 8\u0026ndash;21). The maximal tumor size/burden permitted by our institutional review board is mean tumor diameter 20 mm and 15% of body weight in adult mice (~\u0026thinsp;25 g). The maximal tumor size/burden in our study was not exceeded.\u003c/p\u003e \u003cp\u003e \u003cem\u003eStatistical analysis\u003c/em\u003e \u003c/p\u003e \u003cp\u003eData are presented herein as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of measurement (SEM) from at least three independent experiments. ANOVA, Mann\u0026ndash;Whitney U, or Student\u0026rsquo;s t-test was used when appropriate to perform comparisons. The Kaplan\u0026ndash;Meier method, log-rank test, and multivariate Cox proportional hazard models were used to detect survival differences and identify independent prognostic factors. Statistical analyses were carried out using SPSS 22.0 (SPSS Inc., USA). The level of significance was set at *p\u0026thinsp;\u0026lt;\u0026thinsp;.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;.001, and ****p\u0026thinsp;\u0026lt;\u0026thinsp;.0001.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. ATF4 expression is upregulated in ccRCC and associated with poor outcomes\u003c/h2\u003e\n \u003cp\u003eTo evaluate ATF4 expression and its potential clinical role in ccRCC, we investigated the mRNA expression of ATF4 in ccRCC by analyzing the Cancer Genome Atlas (TCGA) data, indicating that ATF4 expression was significantly higher in ccRCC tumor tissues than in normal tissues (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea). ATF4 expression was also associated with prognosis, and the overall survival (OS) probability of the ATF4 high-expression group was found to be significantly higher than that of the ATF4 low-expression group (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eb). Western blot experiments on five paired ccRCC tissues and normal tissues from patients revealed that ATF4 was upregulated in tumor tissues (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\n \u003cp\u003eWe then evaluated ATF4 expression in ccRCC by conducting an IHC staining analysis of a TMA consisting of 199 ccRCC and 143 normal tissue samples. A variable ATF4 staining intensity was detected in ccRCC tumor tissues using TMAs (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed), and patients were divided into low- and high-expression groups based on the IHC score according to ATF4 expression level. The IHC score for ATF4 in tumor tissues was higher than in normal tissues (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ee). The Kaplan\u0026ndash;Meier analysis revealed that ATF4 high expression was significantly correlated with a shorter OS (p\u0026thinsp;=\u0026thinsp;0.0043; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ef). Consistently, ATF4 expression also correlated with various features of tumor progression, including T stage, M stage, and histological grade. Quantitative analyses based on IHC scores indicated that high ATF4 expression was significantly associated with advanced T stage (T3 and T4), M1 stage, and advanced tumor grade (III and IV; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eg, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Taken together, these findings suggest that ATF4 expression is upregulated in ccRCC and associated with poor outcomes.\u003c/p\u003e\n \u003cp\u003eTable 1 Association of ATF4 expression with clinical and pathological characteristics of 199 ccRCC patients in TMAs.\u003c/p\u003e\n \u003cp\u003e\u003cimg 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\" style=\"width: 1057px;\" width=\"1057\" height=\"916\"\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. ATF4 knockdown attenuates ccRCC cell proliferation, migration, and invasion and induces apoptosis\u003c/h2\u003e\n \u003cp\u003eTo investigate the functional role of ATF4 in ccRCC cells, we first examined the protein expression of ATF4 in normal renal epithelial cells (HEK293T and HKC) and ccRCC cell lines (A498, ACHN, Caki-1, 786-O, and SN12). The results revealed that a higher ATF4 expression was detected in ccRCC cell lines compared with HEK293T and HKC cell lines (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). Among these cell lines, the top two highest expressions of ATF4 were observed in the SN12 and 786-O cell lines, which were then used for further studies in vitro.\u003c/p\u003e\n \u003cp\u003eTwo lentiviral vectors with shRNA against ATF4 (shATF4) were constructed, the efficacy of which was confirmed by Western blot on day 4 after lentivirus infection (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). ATF4 depletion was found to inhibit cell proliferation by MTT assay (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec) and impair migration and invasion abilities by transwell assay (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). Flow cytometry (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee) analyses consistently revealed that ATF4 knockdown induced remarkable early apoptosis in both 786-O and SN12 cells.\u003c/p\u003e\n \u003cp\u003eWe then detected several markers related to mTOR, EMT, and apoptosis by Western blot. ATF4 knockdown was found to exert a remarkable effect on the phosphorylation of S6 and 4EBP1, a readout of mTORC1 signaling. The increased expression of E-cadherin was observed in 786-O cells infected with shATF4, together with the concomitant decreased expression of N-cadherin and vimentin. In addition, shATF4-treated cells exhibited an increase in cleaved caspase-3 activity and a reduction in Bcl-2 expression compared with the NC group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e\n \u003cp\u003eIn vivo, the orthotopic implantation model in BALB/c nude mice was established by transplanting Luc-labelled SN12 cells, stably transfected with NC or shATF4 under the right kidney capsule. On day 21 after injection, tumor signals by bioluminescent imaging and tumor weight in the shATF4 group were significantly lower than in the NC group (Supplementary Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eg). Consistent with the in vitro results, IHC for tumor tissues showed higher Ki-67 and cleaved caspase-3 expressions in shATF4 group (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eh). Altogether, these findings suggest that ATF4 may serve a pro-survival function by promoting the mTOR signaling pathway and EMT and inhibiting apoptosis in ccRCC.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. ATF4 deficiency downregulates NUPR1 expression in ccRCC\u003c/h2\u003e\n \u003cp\u003eTo verify the molecular mechanisms or signaling pathways involved in ATF4-mediated pro-survival in ccRCC, an RNA-seq analysis of 786-O cells was performed after ATF4 knockdown. A heatmap analysis of differentially expressed genes (DEGs, |fold change|\u0026gt;2) showed overall changes (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). A Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis and gene ontology (GO) enrichment analysis were performed to examine the functional role of ATF4 in ccRCC, which indicated that ATF4 may participate in the PI3K/AKT signaling pathway in ccRCC (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). Target genes transcriptionally regulated by ATF4 were acquired based on chromatin immunoprecipitation (ChIP) results from other studies in the database. A Venn diagram of target genes and DEGs was drawn to screen the potential genes, and 75 potential genes were identified and shown in the DEG volcano plot (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec), among which NUPR1 showed the most remarkable fold change (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformatics.com.cn\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe expression correlation between ATF4 and NUPR1 was identified based on ccRCC cases in the TCGA dataset (Supplementary Figure S2a). To verify whether NUPR1 is regulated by ATF4 in ccRCC, the relative mRNA and protein levels of NUPR1 were measured by qRT-PCR and Western blot in 786-O cells and SN12 cells. NUPR1 was found to be downregulated in the shATF4 group (Figs. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef). IHC showed the same results in tumor tissues (Supplementary Figure S2b). Moreover, the linear regression analysis results based on the IHC score for 199 ccRCC tissues by TMAs demonstrated a significant correlation between NUPR1 and ATF4 expressions (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eg). Overall, our data suggest that ATF4 regulates NUPR1 expression during ccRCC progression.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4. NUPR1 expression is correlated with an advanced stage and prognosis in patients with ccRCC\u003c/h2\u003e\n \u003cp\u003eNUPR1 is also known as p8 and is a candidate of metastasis 1 (COM1) and is overexpressed in various cancers in response to several cellular stress factors. Remarkably, multiple types of cancer can be prevented by genetically inactivating NUPR1\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e)\u003c/sup\u003e. Mechanistically, NUPR1 may promote tumor progression by activating the PI3K/AKT signaling pathway, and it is also a critical repressor of ferroptosis that blocks ferroptotic cell death by diminishing iron accumulation and subsequent oxidative damage via LCN2 expression\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eThe data from TCGA revealed that NUPR1 mRNA was significantly upregulated in ccRCC compared with normal tissues and correlated with the tumor clinical stage (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). The OS probability of the NUPR1 high-expression group was significantly lower than the NUPR1 low- and medium-expression groups (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). NUPR1 expression was detected in 199 ccRCC and 143 normal tissues from TMAs via IHC staining. These patients were divided into NUPR1 low- and high-expression groups based on their IHC score according to the variable staining intensity (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). The IHC score for NUPR1 in tumor tissues was markedly higher than the score corresponding to normal tissues (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed). NUPR1 expression was also found to be significantly associated with the T stage (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ee). The Kaplan\u0026ndash;Meier analysis showed that the NUPR1 high-expression group was significantly correlated with shorter OS (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ef). In summary, these findings disclose that NUPR1 expression is upregulated in ccRCC and associated with poor outcomes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5. NUPR1 inhibitor ZZW115 reduces cell proliferation and metastasis and induces apoptosis and ferroptosis\u003c/h2\u003e\n \u003cp\u003eThe NUPR1 inhibitor ZZW115 binds strongly the nuclear localization signal (NLS) of NUPR1, which prevents NUPR1 from translocating to the nucleus. ZZW115 acts mechanistically by mimicking NUPR1 inactivation\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e)\u003c/sup\u003e. To explore whether NUPR1 acts as a tumor promoter and if it can serve as a potential therapeutic target in ccRCC, MTT, and transwell assay were conducted to examine the effect of ZZW115 on ccRCC cell growth, migration, and invasion. In vitro experiments revealed that ZZW115 inhibited cell growth and metastasis and increased the cell death of 786-O and SN12 cells in a dose-dependent manner (Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea, \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb, and Supplementary Figure S3). TUNEL imaging and flow cytometry showed significantly more apoptotic cells in the ZZW115 group than in the control group (Figs. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). The Western blot analysis showed lower expression levels of p-AKT/p-mTOR, N-cad, and vimentin in ZZW115-treated 786-O cells than DMSO and increased expressions of E-cad and cleaved caspase-3. In addition, the ZZW115 treatment induced the accumulation of lipid hydroperoxide, suggesting a promotion of ferroptosis (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ee). Altogether, these findings suggest that ATF4 knockdown may suppress tumor progression by decreasing NUPR1 expression.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n \u003ch2\u003e3.6. ATF4 knockdown induces ferroptosis by targeting NUPR1\u003c/h2\u003e\n \u003cp\u003eNUPR1 is a critical repressor of ferroptosis, and an NUPR1 inhibitor induces ferroptosis in ccRCC. It was hypothesized that ATF4 knockdown could induce ferroptosis by downregulating NUPR1. Erastin dose-dependently induced cell death in ccRCC cells, which was partially rescued by the ferroptosis inhibitor ferrostatin-1 (Supplementary Figure S4a). Previous studies indicated that ATF4 could also have interaction with erastin-induced stress\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003c/sup\u003e. Indeed, erastin was found to dose-dependently induce feedback upregulation of ATF4 expression, suggesting that ATF4 may be associated with ferroptosis (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea). ATF4 deletion simultaneously downregulated erastin-induced NUPR1 protein expression (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eb) and increased erastin-induced lipid peroxidation and cell death (Supplementary Figures S4b and S4c).\u003c/p\u003e\n \u003cp\u003eTreatment with shATF4 induced an evident decrease in mitochondria volume, a distinct reduction (or disappearance) of mitochondrial cristae, and an increase in membrane density, which are described as morphological features in ferroptotic cells (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec). Flow cytometry analysis demonstrated a remarkable increase in lipid peroxidation in the shATF4 group compared with the NC group, which was rescued by the ferroptosis inhibitor ferrostatin-1 (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ed and \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ee).\u003c/p\u003e\n \u003cp\u003eTo examine whether ATF4 deletion increased Fe\u003csup\u003e2+\u003c/sup\u003e levels, an analysis was conducted with the fluorescence probe FerroOrange. The results revealed a significant increase in the relative intracellular Fe\u003csup\u003e2+\u003c/sup\u003e fluorescence intensity in shATF4-transfected 786-O and Caki-1 cells compared with the NC group (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef and S4d). Likewise, the NUPR1 inhibitor ZZW115 increased the intracellular Fe\u003csup\u003e2+\u003c/sup\u003e levels in Caki-1 cells (Figure S4e). Ferrostatin-1 increased cell viability compared with DMSO in the shATF4 group, partially rescuing cell death by ATF4 deletion (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eg). Collectively, these findings indicate that ATF4 knockdown induces ferroptosis by increasing intracellular Fe\u003csup\u003e2+\u003c/sup\u003e levels by downregulating NUPR1 expression in ccRCC cells.\u003c/p\u003e\n \u003ch2\u003e\u003cem\u003e3.7. ATF4 depletion increases the infiltration of CD4\u003c/em\u003e \u003csup\u003e\u0026nbsp;\u003cem\u003e+\u003c/em\u003e\u0026nbsp;\u003c/sup\u003e \u003cem\u003eand CD8\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e \u003cem\u003eT cells and promotes antitumor immunity in vivo\u003c/em\u003e\u003c/h2\u003e\n \u003cp\u003eTo investigate whether ATF4 deletion promotes an antitumor immune response to inhibit tumor growth and metastasis, murine shATF4 (shATF4-M) for RenCa cells was generated and validated by Western blot and proliferation assays on day 4 after lentivirus infection (Supplementary Figures S5a and S5b). An orthotopic implantation model was established by transplanting RenCa cells with negative control shRNA or shATF4-M into the kidney (Supplementary Figures S5c and S5d). ATF4 knockdown significantly reduced the tumor weight and lung metastasis (Figs. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea and \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb).\u003c/p\u003e\n \u003cp\u003eGenomic analysis has proven that ferroptosis is associated with programmed death-ligand 1 (PD-L1) expression, immune cell infiltration, and immunotherapy sensitivity\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e)\u003c/sup\u003e. Although ferroptosis may be involved in a diverse and complex TME, it remains largely unclear whether ATF4 influences TME and immunotherapy in ccRCC. The TME alterations were detected in the BALB/c mice, and intratumor infiltration of CD4\u003csup\u003e\u0026minus;\u003c/sup\u003e, CD8\u003csup\u003e\u0026minus;\u003c/sup\u003e, and granzyme B\u003csup\u003e+\u003c/sup\u003e cells remarkably increased in tumors from the shATF4 group (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ec). The same tendency was observed in tumor tissues from TMAs (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ed).\u003c/p\u003e\n \u003cp\u003eRecent studies have proven that CD8\u003csup\u003e+\u003c/sup\u003e T cells suppress tumor growth by inducing ferroptosis. Targeting tumor ferroptosis-associated metabolism may thus improve the efficacy of cancer immunotherapy. A synergistic effect has been reported between PD-L1 blockade antibodies and ferroptosis activators, which suppress tumor growth both in vitro and in vivo\u003csup\u003e(\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e)\u003c/sup\u003e. The effect of ATF4 deletion inducing ferroptosis suggests a treatment strategy of combining immunotherapy with ATF4 pathway inhibition. In this study, to assess the efficacy of the combinatorial therapy, xenograft tumors transplanted with RenCa cells were treated with ZZW115, anti-PD1, or both. The weight of combinatorial-treated tumors was found to be significantly decreased compared with either tumors treated with DMSO or a single agent (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ee). Consistent with ATF4 deletion, treatment with ZZW115 remarkably promoted CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration in vivo (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ef). Collectively, our data indicate that besides impairing proliferation and motility and enhancing apoptosis and ferroptosis in tumor cells, ATF4 deletion may increase CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration and promote antitumor immunity to suppress tumor growth in vivo, indicating the potential value of targeting ATF4 in treating ccRCC.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDuring tumor development, cancer cells encounter various stressful environmental conditions, such as hypoxia, nutrition deprivation, and the effect of drugs\u003csup\u003e(\u003cspan additionalcitationids=\"CR39 CR40\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e)\u003c/sup\u003e. Several proteins are activated to initiate adaptive pathways in response to stress. ATF4, a stress-induced transcription factor, is usually elevated to regulate its target genes exerting corresponding functions, and this facilitates cancer cell survival, growth, development, and progression\u003csup\u003e(\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, ATF4 knockdown was found to diminish ccRCC cell proliferation, migration, and invasion by suppressing mTOR activity and the EMT pathway (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef). Our data also suggest a potentially negative role of ATF4 in ferroptosis, and ATF4 deletion triggered ferroptosis by increasing Fe\u003csup\u003e2+\u003c/sup\u003e accumulation in ccRCC (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Reports have described how ATF4 promotes ER stress-induced cell death by targeting downstream genes\u003csup\u003e(\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e)\u003c/sup\u003e, and in some contexts, ATF4 has been found to induce a pro-apoptotic effect by dimerizing with C/EBP homologous protein (CHOP) \u003csup\u003e(\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e)\u003c/sup\u003e. Interestingly, in our study, ATF4 knockdown promoted apoptosis in ccRCC cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), which suggests that ATF4 plays an anti-apoptotic function in a CHOP-independent manner. It was hypothesized that ATF4 deletion could increase vulnerability to cellular stress during tumor growth, thereby resulting in the indirect induction of cell death. Consistently, inhibiting cell growth by ATF4 deletion was partially rescued by ferrostatin-1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eg). The transcriptional regulation of ATF4 is, therefore, complex, and multiple pathways may be involved in ATF4-mediated cell biological processes. ATF4 exhibits a dual functionality within tumor cells\u003csup\u003e(\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e)\u003c/sup\u003e. On the one hand, it regulates genes associated with amino acid transportation and metabolism, protection against oxidative stress, and maintenance of protein homeostasis\u003csup\u003e(\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e)\u003c/sup\u003e. On the other hand, ATF4 can trigger apoptosis, cell-cycle arrest, and senescence\u003csup\u003e(\u003cspan additionalcitationids=\"CR51 CR52 CR53 CR54 CR55\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e)\u003c/sup\u003e. ATF4 induces GADD34, leading to a negative feedback loop involving dephosphorylation of eIF2a and restoration of translation\u003csup\u003e(\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e)\u003c/sup\u003e. ATF4 exerts a dual effect on mTOR activity, whereby it promotes amino acid availability through autophagy and concurrently upregulates adaptive genes. Conversely, ATF4 also induces the expression of mTOR repressors, namely, SESN2, DDIT4, and REDD1\u003csup\u003e(\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e)\u003c/sup\u003e. The ATF4-initiated transcriptional program concurrently diminishes the expression of the anti-apoptotic BCL2 protein while augmenting pro-apoptotic signaling mediated by the proteins BIM, NOXA, and PUMA\u003csup\u003e(\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e)\u003c/sup\u003e. Consequently, the outcome of ATF4 activation is highly context-dependent.\u003c/p\u003e \u003cp\u003eBased on previous studies and the results obtained in this study, we conclude that NUPR1 is a potential key target gene of ATF4 and serves as a predictor of ccRCC\u003csup\u003e(\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e)\u003c/sup\u003e. ATF4 and NUPR1 were upregulated in ccRCC tissues and significantly associated with poor outcomes (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ef, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), and the NUPR1 inhibitor, ZZW115, suppressed ccRCC tumor growth in vitro and in vivo (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). In addition to the regulation of apoptosis, it was identified that ATF4 is required for NUPR1 expression in response to ferroptosis activators in ccRCC. In some cases, acquired sensitivity to ferroptosis has been attributed to the specific metabolic reprogramming of cancer cells, and this provides new opportunities for treating tumors that are insensitive to regular therapy. Currently, triggering ferroptosis is a promising approach for inducing tumor cell death, especially in tumors resistant to traditional treatment strategies\u003csup\u003e(\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNUPR1, a stress-induced protein, engages in a positive feedback loop with ATF4. NUPR1 was found to present nuclear localization complexed to other protein(s) or nucleic acids in sub-confluent cells but localized throughout the whole cell in those grown to high density or an arrested Go state of the cells, indicating the nuclear import and export of NUPR1. It is reported that NUPR1 exists in the cell as part of protein complexes and directs their subcellular localization to regulate the protein function\u003csup\u003e(\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e)\u003c/sup\u003e. An architectural role in transcription, already proposed for NUPR1, is compatible with nuclear localization\u003csup\u003e(\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e)\u003c/sup\u003e. ATF4 is confirmed to regulate NUPR1 transcriptionally\u003csup\u003e(\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e)\u003c/sup\u003e. As our results confirm, NUPR1 and ATF4 present a nuclear localization, suggesting the function of transcription. However, a cytoplasmic localization of NUPR1 is reported in papillary carcinomas, which might reflect disease progression. Further investigations must be conducted to uncover the molecular mechanism of difference in the localization of NUPR1 based on cell density, environmental changes, tumor type, and other factors.\u003c/p\u003e \u003cp\u003eNevertheless, our data support that ATF4/NUPR1 axis plays a pro-survival role by activating the AKT/mTOR pathway and decreasing the apoptosis and ferroptosis cell death in ccRCC. Furthermore, the current study provides evidence that targeting ATF4/NUPR1 with or without a ferroptosis inducer may be a new effective treatment for ccRCC.\u003c/p\u003e \u003cp\u003eFerroptotic cancer cells can release a set of immunostimulatory signals, allowing immune cells to properly locate the site of dying tumor cells\u003csup\u003e(\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e)\u003c/sup\u003e. Several studies have demonstrated that ferroptosis inducers could be associated with TME features and enhance the efficacy of immune checkpoint inhibitor immunotherapy\u003csup\u003e(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e)\u003c/sup\u003e. Immunostimulatory signals released by ferroptotic cancer cells can promote the phenotypic maturation of dendritic cells and increase the efficiency of macrophages, particularly M1-like tumor-associated macrophages, in the phagocytosis of ferroptotic cancer cells, strengthening CD8\u003csup\u003e+\u003c/sup\u003e T cell-mediated tumor suppression. Interferon-γ secreted by CD8\u003csup\u003e+\u003c/sup\u003e T cells represses solute carrier family 7 member 11 (SLC7A11) expression in cancer cells, which promotes cancer cell ferroptosis. In addition, the induction of ferroptosis can impair regulatory T (Treg) cells, myeloid-derived suppressor cells (MDSCs), and M2-like TAMs, augmenting antitumor immunity\u003csup\u003e(\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e)\u003c/sup\u003e. Memory CD4\u003csup\u003e+\u003c/sup\u003e T cells and M0 macrophages can be activated via ferroptosis-related lncRNA in hepatocellular carcinoma\u003csup\u003e(\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e)\u003c/sup\u003e. Our study consistently confirmed, in vivo, that activated CD8\u003csup\u003e+\u003c/sup\u003e T cells enhanced ferroptosis-specific lipid peroxidation in tumor cells. In turn, increased ferroptosis contributes to the antitumor efficacy of immunotherapy. Therefore, targeting the tumor ferroptosis pathway would be a therapeutic approach to use in combination with checkpoint blockade \u003csup\u003e(\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e)\u003c/sup\u003e. The present study confirms that ATF4 deletion and NUPR1 inhibitor treatment increased CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cell infiltration, which is presumably a result of ferroptosis. However, additional studies are required to identify these mechanisms experimentally.\u003c/p\u003e \u003cp\u003eTherapeutic interventions can also contribute to a stressed state\u003csup\u003e(\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e)\u003c/sup\u003e. Unsurprisingly, ATF4 mediates resistance to therapeutic agents in various cancers. NUPR1 expression is consistently associated with resistance to several drugs\u003csup\u003e(\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e)\u003c/sup\u003e. For example, NUPR1 silencing reverses sorafenib resistance in ccRCC by targeting the PTEN/AKT/mTOR pathway \u003csup\u003e(\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e)\u003c/sup\u003e. Therefore, suppressing the ATF4/NUPR1 pathway may reduce the capacity for drug-induced stress in tumor cells, resulting in cell death induction, proliferation inhibition, and increased therapeutic efficacy. The potential beneficial effects of NUPR1 inhibitors in combination with anti-PD1 in ccRCC were confirmed via animal experiments.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eATF4 knockdown inhibits proliferation and motility and induces apoptosis and ferroptosis via NUPR1 in ccRCC, increasing the infiltration of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells and enhancing antitumor immunity. Therefore, targeting ATF4/NUPR1 may be a potential alternative therapeutic strategy for controlling ccRCC progression. Further studies are required to elucidate the mechanism whereby ATF4 exerts antitumor immunity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;Data Availability Statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available in the methods and/or supplementary material of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXin Ma, Yan Huang, Hongzhao Li and Yongliang Lu conceived the project and designed the experiments. Weihao Chen and Yundong Xuan performed the cell experiments.Yongliang Lu, Yundong Xuan, Shengpan Wu and Hanfeng Wang conducted the plasmid construction and molecular experiments. Tao Guo, Chenfeng Wang and Shuo Tian provided the human tissue samples and clinical information. Yongliang Lu and Yundong Xuan performed the immunofluorescence assays. Yongliang Lu, Weihao Chen, Shengpan Wu, Xupeng zhao, Wenlei Zhao and Tao Guo performed the IHC and H\u0026amp;E staining. Yongliang Lu, Weihao Chen, Yundong Xuan, Shengpan Wu, Huaikang Li, Dong Lai and Tao Guo performed the animal experiments. Yongliang Lu, Xiubin Li and Xing Huang performed the bioinformatics analyses and conducted the RNA sequencing analysis. Yongliang Lu Weihao chen and Yan Huang analyzed and interpreted the data. Yan Huang, Hongzhao Li and Xin Ma supervised the overall execution of the experiments. The manuscript was written by Yongliang Lu with support from Baojun Wang, Xu Zhang, Yan Huang, Hongzhao Li and Xin Ma. All authors have read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Research Ethics Committee of the PLA General Hospital. The animal study protocol was approved by the Institutional Animal Care and Use Committee of the PLA General Hospital. The study is reported in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patients to publish this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFerlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Pi\u0026ntilde;eros M, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144(8):1941\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSiegel RL, Miller KD, Fuchs HE, Jemal A, Cancer Statistics. 2021. CA Cancer J Clin. 2021;71(1):7\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohen HT, McGovern FJ. Renal-cell carcinoma. N Engl J Med. 2005;353(23):2477\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicketts CJ, Crooks DR, Sourbier C, Schmidt LS, Srinivasan R, Linehan WM. SnapShot: Renal Cell Carcinoma. Cancer Cell. 2016;29(4):610\u0026ndash;e1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLjungberg B, Albiges L, Abu-Ghanem Y, Bedke J, Capitanio U, Dabestani S et al. European Association of Urology Guidelines on Renal Cell Carcinoma: The 2022 Update. Eur Urol. 2022.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDejeans N, Barroso K, Fernandez-Zapico ME, Samali A, Chevet E. Novel roles of the unfolded protein response in the control of tumor development and aggressiveness. Semin Cancer Biol. 2015;33:67\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaurel M, McGrath EP, Mnich K, Healy S, Chevet E, Samali A. Controlling the unfolded protein response-mediated life and death decisions in cancer. Semin Cancer Biol. 2015;33:57\u0026ndash;66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUrra H, Dufey E, Avril T, Chevet E, Hetz C. Endoplasmic Reticulum Stress and the Hallmarks of Cancer. Trends Cancer. 2016;2(5):252\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSullivan LB, Chandel NS. Mitochondrial reactive oxygen species and cancer. Cancer Metab. 2014;2:17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKilberg MS, Shan J, Su N. ATF4-dependent transcription mediates signaling of amino acid limitation. Trends Endocrinol Metab. 2009;20(9):436\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuhr M, Torgersen ML, Szalai P, Hashim A, Brech A, Staerk J, et al. The kinase PERK and the transcription factor ATF4 play distinct and essential roles in autophagy resulting from tunicamycin-induced ER stress. J Biol Chem. 2019;294(20):8197\u0026ndash;217.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRozpedek W, Pytel D, Mucha B, Leszczynska H, Diehl JA, Majsterek I. The Role of the PERK/eIF2α/ATF4/CHOP Signaling Pathway in Tumor Progression During Endoplasmic Reticulum Stress. Curr Mol Med. 2016;16(6):533\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu M, Wang L, Xia X, Wu Y, Zhu C, Duan M, et al. Regulated lytic cell death in breast cancer. Cell Biol Int. 2022;46(1):12\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang C, Zhang X, Yang M, Dong X. Recent Progress in Ferroptosis Inducers for Cancer Therapy. Adv Mater. 2019;31(51):e1904197.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee AS. Glucose-regulated proteins in cancer: molecular mechanisms and therapeutic potential. Nat Rev Cancer. 2014;14(4):263\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun X, Ou Z, Xie M, Kang R, Fan Y, Niu X, et al. HSPB1 as a novel regulator of ferroptotic cancer cell death. Oncogene. 2015;34(45):5617\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBai D, Feng H, Yang J, Yin A, Lin X, Qian A, et al. Genomic analysis uncovers prognostic and immunogenic characteristics of ferroptosis for clear cell renal cell carcinoma. Mol Ther Nucleic Acids. 2021;25:186\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao Y, Li H, Ma X, Fan Y, Ni D, Zhang Y, et al. KLF6 Suppresses Metastasis of Clear Cell Renal Cell Carcinoma via Transcriptional Repression of E2F1. Cancer Res. 2017;77(2):330\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamanishi J, Mandai M, Iwasaki M, Okazaki T, Tanaka Y, Yamaguchi K, et al. Programmed cell death 1 ligand 1 and tumor-infiltrating CD8\u0026thinsp;+\u0026thinsp;T lymphocytes are prognostic factors of human ovarian cancer. Proc Natl Acad Sci U S A. 2007;104(9):3360\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorikawa N, Abiko K, Matsumura N, Hamanishi J, Baba T, Yamaguchi K, et al. Expression of Vascular Endothelial Growth Factor in Ovarian Cancer Inhibits Tumor Immunity through the Accumulation of Myeloid-Derived Suppressor Cells. Clin Cancer Res. 2017;23(2):587\u0026ndash;99.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRao X, Huang X, Zhou Z, Lin X. An improvement of the 2ˆ(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath. 2013;3(3):71\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSandi MJ, Hamidi T, Malicet C, Cano C, Loncle C, Pierres A, et al. p8 expression controls pancreatic cancer cell migration, invasion, adhesion, and tumorigenesis. J Cell Physiol. 2011;226(12):3442\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEmma MR, Iovanna JL, Bachvarov D, Puleio R, Loria GR, Augello G, et al. NUPR1, a new target in liver cancer: implication in controlling cell growth, migration, invasion and sorafenib resistance. Cell Death Dis. 2016;7(6):e2269.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo X, Wang W, Hu J, Feng K, Pan Y, Zhang L, et al. Lentivirus-mediated RNAi knockdown of NUPR1 inhibits human nonsmall cell lung cancer growth in vitro and in vivo. Anat Rec (Hoboken). 2012;295(12):2114\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim KS, Jin DI, Yoon S, Baek SY, Kim BS, Oh SO. Expression and roles of NUPR1 in cholangiocarcinoma cells. Anat Cell Biol. 2012;45(1):17\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi J, Ren S, Liu Y, Lian Z, Dong B, Yao Y, et al. Knockdown of NUPR1 inhibits the proliferation of glioblastoma cells via ERK1/2, p38 MAPK and caspase-3. J Neurooncol. 2017;132(1):15\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeng C, Li X, Li A, Yi B, Peng X, Huang X, et al. Knockdown of NUPR1 inhibits the growth of U266 and RPMI8226 multiple myeloma cell lines via activating PTEN and caspase activation\u0026ndash;dependent apoptosis. Oncol Rep. 2018;40(3):1487\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou C, Xu J, Lin J, Lin R, Chen K, Kong J, et al. Long Noncoding RNA FEZF1-AS1 Promotes Osteosarcoma Progression by Regulating the miR-4443/NUPR1 Axis. Oncol Res. 2018;26(9):1335\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu J, Zhu H, Li R, Jiang Q, Luan W, Shi J, et al. Oncogenic Role of NUPR1 in Ovarian Cancer. Onco Targets Ther. 2020;13:12289\u0026ndash;300.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang L, Wang W, Li Z, Zhao Y, Qin Z. NUPR1 participates in YAP-mediate gastric cancer malignancy and drug resistance via AKT and p21 activation. J Pharm Pharmacol. 2021;73(6):740\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang K, Zhang Z, Bai Z, Ma X, Guo W, Wang Y. Enhancement of gemcitabine sensitivity in pancreatic cancer by co-regulation of dCK and p8 expression. Oncol Rep. 2011;25(4):963\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang W, Zhang C, Zhang X, Sun L, Li J, Zuo J. CircRNA HIPK3 promotes the progression of oral squamous cell carcinoma through upregulation of the NUPR1/PI3K/AKT pathway by sponging miR-637. Ann Transl Med. 2021;9(10):860.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Song X, Kuang F, Zhang Q, Xie Y, Kang R, et al. NUPR1 is a critical repressor of ferroptosis. Nat Commun. 2021;12(1):647.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLan W, Santofimia-Casta\u0026ntilde;o P, Swayden M, Xia Y, Zhou Z, Audebert S et al. ZZW-115-dependent inhibition of NUPR1 nuclear translocation sensitizes cancer cells to genotoxic agents. JCI Insight. 2020;5(18).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang S, Chen S, Ying Y, Ma X, Shen H, Li J, et al. Comprehensive Analysis of Ferroptosis Regulators With Regard to PD-L1 and Immune Infiltration in Clear Cell Renal Cell Carcinoma. Front Cell Dev Biol. 2021;9:676142.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang W, Green M, Choi JE, Gij\u0026oacute;n M, Kennedy PD, Johnson JK, et al. CD8(+) T cells regulate tumour ferroptosis during cancer immunotherapy. Nature. 2019;569(7755):270\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang R, Xu J, Zhang B, Liu J, Liang C, Hua J, et al. Ferroptosis, necroptosis, and pyroptosis in anticancer immunity. J Hematol Oncol. 2020;13(1):110.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarris CS, Pittet MJ. ER Stress in Dendritic Cells Promotes Cancer. Cell. 2015;161(7):1492\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTodoric J, Antonucci L, Di Caro G, Li N, Wu X, Lytle NK, et al. Stress-Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas. Cancer Cell. 2017;32(6):824\u0026ndash;39e8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLinares JF, Cordes T, Duran A, Reina-Campos M, Valencia T, Ahn CS, et al. ATF4-Induced Metabolic Reprograming Is a Synthetic Vulnerability of the p62-Deficient Tumor Stroma. Cell Metab. 2017;26(6):817\u0026ndash;29e6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeton-Rogers S, Oncogenes. Coping with stress. Nat Rev Cancer. 2017;17(2):76\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArmstrong JL, Flockhart R, Veal GJ, Lovat PE, Redfern CP. Regulation of endoplasmic reticulum stress-induced cell death by ATF4 in neuroectodermal tumor cells. J Biol Chem. 2010;285(9):6091\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang H, Hua X, Liu N, Li X, Liu S, Chen X, et al. Anacardic acid induces cell apoptosis associated with induction of ATF4-dependent endoplasmic reticulum stress. Toxicol Lett. 2014;228(3):170\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan J, Back SH, Hur J, Lin YH, Gildersleeve R, Shan J, et al. ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat Cell Biol. 2013;15(5):481\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsumoto H, Miyazaki S, Matsuyama S, Takeda M, Kawano M, Nakagawa H, et al. Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression. Biol Open. 2013;2(10):1084\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeske BF, Fusakio ME, Zhou D, Shan J, McClintick JN, Kilberg MS, et al. CHOP induces activating transcription factor 5 (ATF5) to trigger apoptosis in response to perturbations in protein homeostasis. Mol Biol Cell. 2013;24(15):2477\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWortel IMN, van der Meer LT, Kilberg MS, van Leeuwen FN. Surviving Stress: Modulation of ATF4-Mediated Stress Responses in Normal and Malignant Cells. Trends Endocrinol Metab. 2017;28(11):794\u0026ndash;806.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M, et al. An Integrated Stress Response Regulates Amino Acid Metabolism and Resistance to Oxidative Stress. Mol Cell. 2003;11(3):619\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNovoa I, Zhang Y, Zeng H, Jungreis R, Harding HP, Ron D. Stress-induced gene expression requires programmed recovery from translational repression. Embo j. 2003;22(5):1180\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFrank CL, Ge X, Xie Z, Zhou Y, Tsai LH. Control of activating transcription factor 4 (ATF4) persistence by multisite phosphorylation impacts cell cycle progression and neurogenesis. J Biol Chem. 2010;285(43):33324\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan J, Back SH, Hur J, Lin Y-H, Gildersleeve R, Shan J, et al. ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat Cell Biol. 2013;15(5):481\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin H-K, Chen Z, Wang G, Nardella C, Lee S-W, Chan C-H, et al. Skp2 targeting suppresses tumorigenesis by Arf-p53-independent cellular senescence. Nature. 2010;464(7287):374\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHiramatsu N, Messah C, Han J, Lavail M, Lin J. Translational and post-translational regulation of XIAP by eIF2α and ATF4 promotes ER stress๊nduced cell death during the unfolded protein response (151.5). FASEB J. 2014;28:1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsumoto H, Miyazaki S, Matsuyama S, Takeda M, Kawano M, Nakagawa H, et al. Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression. Biology Open. 2013;2(10):1084\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhoka N, Yoshii S, Hattori T, Onozaki K, Hayashi H. TRB3, a novel ER stress-inducible gene, is induced via ATF4\u0026ndash;CHOP pathway and is involved in cell death. EMBO J. 2005;24(6):1243\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshizawa J, Kojima K, Chachad D, Ruvolo P, Ruvolo V, Jacamo RO, et al. ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies. Sci Signal. 2016;9(415):ra17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan B-J, Krokowski D, Majumder M, Schmotzer CL, Kimball SR, Merrick WC, et al. Translational Control during Endoplasmic Reticulum Stress beyond Phosphorylation of the Translation Initiation Factor eIF2α*. J Biol Chem. 2014;289(18):12593\u0026ndash;611.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu X, Long YC. Autophagy modulates amino acid signaling network in myotubes: differential effects on mTORC1 pathway and the integrated stress response. FASEB J. 2015;29(2):394\u0026ndash;407.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDennis MD, McGhee NK, Jefferson LS, Kimball SR. Regulated in DNA damage and development 1 (REDD1) promotes cell survival during serum deprivation by sustaining repression of signaling through the mechanistic target of rapamycin in complex 1 (mTORC1). Cell Signal. 2013;25(12):2709\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol. 2012;13(2):89\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSzegezdi E, Logue SE, Gorman AM, Samali A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO Rep. 2006;7(9):880\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe W, Cheng F, Zheng B, Wang J, Zhao G, Yao Z, et al. NUPR1 is a novel potential biomarker and confers resistance to sorafenib in clear cell renal cell carcinoma by increasing stemness and targeting the PTEN/AKT/mTOR pathway. Aging. 2021;13(10):14015\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriedmann Angeli JP, Krysko DV, Conrad M. Ferroptosis at the crossroads of cancer-acquired drug resistance and immune evasion. Nat Rev Cancer. 2019;19(7):405\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValacco MP, Varone C, Malicet C, C\u0026aacute;nepa E, Iovanna JL, Moreno S. Cell growth-dependent subcellular localization of p8. J Cell Biochem. 2006;97(5):1066\u0026ndash;79.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHoffmeister A, Ropolo A, Vasseur S, Mallo GV, Bodeker H, Ritz-Laser B, et al. The HMG-I/Y-related protein p8 binds to p300 and Pax2 trans-activation domain-interacting protein to regulate the trans-activation activity of the Pax2A and Pax2B transcription factors on the glucagon gene promoter. J Biol Chem. 2002;277(25):22314\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin HO, Seo SK, Woo SH, Choe TB, Hong SI, Kim JI, et al. Nuclear protein 1 induced by ATF4 in response to various stressors acts as a positive regulator on the transcriptional activation of ATF4. IUBMB Life. 2009;61(12):1153\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYu B, Choi B, Li W, Kim DH. Magnetic field boosted ferroptosis-like cell death and responsive MRI using hybrid vesicles for cancer immunotherapy. Nat Commun. 2020;11(1):3637.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLei G, Zhuang L, Gan B. Targeting ferroptosis as a vulnerability in cancer. Nat Rev Cancer. 2022;22(7):381\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Z, Peng B, Liang Q, Chen X, Cai Y, Zeng S, et al. Construction of a Ferroptosis-Related Nine-lncRNA Signature for Predicting Prognosis and Immune Response in Hepatocellular Carcinoma. Front Immunol. 2021;12:719175.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagelkerke A, Bussink J, Sweep FC, Span PN. The unfolded protein response as a target for cancer therapy. Biochim Biophys Acta. 2014;1846(2):277\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalicet C, Giroux V, Vasseur S, Dagorn JC, Neira JL, Iovanna JL. Regulation of apoptosis by the p8/prothymosin alpha complex. Proc Natl Acad Sci U S A. 2006;103(8):2671\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ccRCC, ATF4, NUPR1, Ferroptosis, Immunosuppression","lastPublishedDoi":"10.21203/rs.3.rs-3870049/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3870049/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCancer cells encounter unavoidable stress during tumor growth. The stress-induced transcription factor, activating transcription factor 4 (ATF4), has been reported to upregulate various adaptive genes involved in salvage pathways to alleviate stress and promote tumor progression. However, this effect is unknown in clear cell renal cell carcinoma (ccRCC). In this study, we found that ATF4 expression was remarkably upregulated in tumor tissues and associated with poor ccRCC outcomes. ATF4 depletion significantly impaired ccRCC cell proliferation, migration, and invasion in vitro and in vivo by inhibiting the AKT/mTOR and epithelial\u0026ndash;mesenchymal transition (EMT)-related signaling pathway. RNA sequencing and functional studies identified nuclear protein 1 (NUPR1) as a key downstream target of ATF4 for repressing ferroptosis and promoting ccRCC cell survival. In addition, targeting ATF4 or pharmacological inhibition using NUPR1 inhibitor ZZW115 promoted antitumor immunity in syngeneic graft mouse models, represented by increased infiltration of CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells. Furthermore, ZZW115 could improve the response to the PD-1 immune checkpoint blockade. The results demonstrate that the ATF4/NUPR1 signaling axis promotes ccRCC survival and facilitates tumor-mediated immunosuppression, providing a set of potential targets and prognostic indicators for ccRCC patients.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"ATF4/NUPR1 axis promotes cancer cell survival and mediates immunosuppression in clear cell renal cell carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-20 18:07:03","doi":"10.21203/rs.3.rs-3870049/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-14T04:06:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-13T17:51:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217786533781063385292361703650057453523","date":"2024-08-09T12:49:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-17T14:19:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"173396272431299350954955995165555966133","date":"2024-07-12T13:34:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-28T16:32:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-16T13:44:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-16T13:40:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Oncology","date":"2024-01-16T14:02:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"94ec7184-d7c9-4a6c-826b-ab926817ab4a","owner":[],"postedDate":"February 20th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-10-22T06:53:41+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-20 18:07:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3870049","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3870049","identity":"rs-3870049","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}